Methods for conducting multiplexed assays

ABSTRACT

The invention relates to methods for conducting solid-phase binding assays. One example is an assay method having improved analyte specificity where specificity is limited by the presence of non-specific binding interactions.

CROSS REFERENCE TO RELATED APPLICATIONS

The present application is a continuation of copending application Ser.No. 14/847,761, filed on Sep. 8, 2015, which claims the benefit of U.S.Provisional Application No. 62/047,097, filed Sep. 8, 2014, the entirecontents of which are incorporated herein by reference. Reference ismade to U.S. Provisional Application Ser. Nos. 61/775,860 and61/778,727, filed Mar. 11, 2013 and Mar. 13, 2013, respectively, thedisclosures of each are incorporated herein by reference in itsentirety. Reference is also made to the following U.S. applications62/013,823, 61/993,581; Ser. No. 14/206,284; Ser. No. 14/208,040, andSer. No. 14/203,638, the disclosures of which are hereby incorporatedherein by reference.

INCORPORATION BY REFERENCE OF SEQUENCE LISTING

The Sequence Listing in the ASCII text file, named as31380_SequenceListing.txt of 10 KB, created on Sep. 8, 2015, andsubmitted to the United States Patent and Trademark Office via EFS-Web,is incorporated herein by reference.

FIELD OF THE INVENTION

Improved methods and products for conducting binding assays areprovided. These methods include the use of a linking agent complex thatenables the user to configure an assay to meet his/her needs from astandard set of assay materials. The products and methods of theinvention greatly enhance productivity and flexibility in assaydevelopment.

BACKGROUND OF THE INVENTION

A substantial body of literature has been developed concerningtechniques that employ binding reactions, e.g., antigen-antibodyreactions, nucleic acid hybridization and receptor-ligand reactions, forthe sensitive measurement of analytes of interest in samples. The highdegree of specificity in many biochemical binding systems has led tomany assay methods and systems of value in a variety of marketsincluding basic research, human and veterinary diagnostics,environmental monitoring and industrial testing. The presence of ananalyte of interest may be measured by directly measuring theparticipation of the analyte in a binding reaction. In some approaches,this participation may be indicated through the measurement of anobservable label attached to one or more of the binding materials.

Commercially available assays are generally supplied in a pre-setconfiguration, offering the user little or no flexibility to evaluatetarget(s) that may be of unique interest to him or her. Such commercialpanels may include target analytes that are of little or no interestand/or they may not include the desired target analyte(s). Therefore,there is a need to provide the user a flexible method to configure auser-defined multiplexed assay using a standard set of assay materialsand methods.

SUMMARY OF THE INVENTION

The present invention contemplates the following specific embodiments.Various modifications, additions and alterations may be made toembodiments described herein by one skilled in the art without departingfrom the spirit and scope of the invention. Such modifications,additions, and alterations are intended to fall within the scope of theclaims.

Embodiment (1): a method of conducting a multiplexed binding assay for aplurality of analytes of interest comprising:

combining, in one or more steps, the following components: a samplecomprising a first analyte of interest and a second analyte of interest,a first targeting agent immobilized on a first binding domain, a firsttargeting agent complement connected to a linking agent, wherein thefirst targeting agent complement is a binding partner of the firsttargeting agent, a first binding reagent connected to a supplementallinking agent, wherein the first binding reagent is a binding partner ofthe first analyte, a second targeting agent immobilized on a secondbinding domain, a second targeting agent complement connected to alinking agent, wherein the second targeting agent complement is abinding partner of the second targeting agent, a second binding reagentconnected to a supplemental linking agent, wherein the second bindingreagent is a binding partner of the second analyte, and optionally, atleast two copies of a bridging agent, wherein, if the bridging agent isomitted, each linking agent is a binding partner of the supplementallinking agent, or if the bridging agent is included, the bridging agenthas a first binding site for one of the linking agents and an additionalbinding site for one of the supplemental linking agents;

forming: a first binding complex on the first binding domain comprisingthe first targeting agent, the first targeting agent complement, thefirst binding reagent and the first analyte, and a second bindingcomplex on the second binding domain comprising the second targetingagent, the second targeting agent complement, the second binding reagentand the second analyte, and

measuring the amount of the first and second analytes on the first andsecond binding domains, respectively.

In one example of embodiment (1), referred to as embodiment (1)(a), thesample contains one or more additional analytes of interest and for eachadditional analyte of interest, the combining step further comprisescombining, in one or more steps, an additional targeting agentimmobilized on an additional binding domain, an additional targetingagent complement connected to a linking agent, and an additional bindingreagent connected to a supplemental linking agent, and an additionalbinding complex on the additional binding domain comprising theadditional targeting agent, the additional targeting agent complement,the additional binding reagent and the additional analyte; the formingstep further comprises forming an additional binding complex on theadditional binding domain comprising the additional targeting agent, theadditional targeting agent complement, the additional binding reagentand the additional analyte; and the measurement further comprisesmeasuring the amount of the additional analyte on the additional bindingdomain.

In a further example of embodiment (1), referred to as embodiment(1)(b), the first targeting agent complement and the first bindingreagent are provided as a pre-bound first targeting complex comprisingthe first targeting agent complement and the first binding reagentlinked through a binding interaction between the linking agent andsupplemental linking agent; and the second targeting agent complementand the second binding reagent are provided as a pre-bound secondtargeting complex comprising the second targeting agent complement andthe second binding reagent linked through a binding interaction betweenthe linking agent and supplemental linking agent.

Moreover, in another example of embodiment (1), referred to asembodiment (1)(c), the first targeting complex is provided pre-bound tothe first targeting agent immobilized on the first binding domain; andthe second targeting complex is provided pre-bound to the secondtargeting agent immobilized on the second binding domain. In thisexample, combining step further includes: combining the first and secondtargeting complexes with the sample to form a mixture thereof, bindingthe first analyte to the first binding reagent in the first targetingcomplex and binding the second analyte to the second binding reagent inthe second targeting complex, contacting a mixture of the first andsecond targeting complexes bound to first and second analytes,respectively, with the first and second binding domains; and binding thefirst targeting complex to the first targeting agent on the firstbinding domain and binding the second targeting complex to the secondtargeting agent on the second binding domain.

In particular example of embodiment (1), referred to as embodiment(1)(d), the combining step further includes combining, in a first volumeof liquid, said first targeting agent complement, said first bindingreagent and, if used, said bridging reagent and linking said firsttargeting agent complement and said first binding reagent through theirattached linking agents to form a first targeting complex; andcombining, in a second volume of liquid, said second targeting agentcomplement, said second binding reagent and, if used, said bridgingreagent and linking said second targeting agent complex complement andsaid second binding reagent through their attached linking agents toform a second targeting complex.

In a specific example of embodiment (1), referred to as embodiment(1)(e), the combining step further includes combining said first andsecond targeting complexes, contacting the combination of said first andsecond targeting complexes with said first and second binding domains,and binding said first targeting complex to said first targeting agenton said first binding domain and binding said second targeting complexto said second targeting agent on said second binding domain. In thisexample, the combining step further includes: combining the first andsecond targeting complexes with the sample to form a mixture thereof,binding the first analyte to the first binding reagent in the firsttargeting complex and binding the second analyte to the second bindingreagent in the second targeting complex, contacting a mixture of thefirst and second targeting complexes bound to first and second analytes,respectively, with the first and second binding domains; and binding thefirst targeting complex to the first targeting agent on the firstbinding domain and binding the second targeting complex to the secondtargeting agent on the second binding domain.

In embodiments (1)(c) and (1)(e), the first and second targetingcomplexes can be combined with the sample prior to contacting the firstand second targeting complexes with the first and second bindingdomains. Moreover, in these specific embodiments, the first and secondtargeting complexes can be combined with the sample after contacting thefirst and second targeting complexes with the first and second bindingdomains; or the first and second targeting complexes can be combinedwith the sample and contacted with the first and second binding domainsat the same time.

In embodiment (1)(d), the combining step can further include the stepsof: contacting said first and second targeting complexes on said firstand second binding domains with said sample, binding said first analyteto the first binding reagent in said first targeting complex and bindingsaid second analyte to said second binding reagent in said secondtargeting complex.

In embodiments (1) and (1)(a)-(e), the bridging agent can be omitted andthe linking agent can be bound to the supplemental linking agent througha binding interaction between (a) a thiol group and a maleimide oriodoacetamide groups; (b) an aldehyde and a hydrazide; or (c) an alkyneand an azide.

In embodiments (1) and (1)(a)-(e), the bridging agent can be omitted and(a) the linking agent is biotin and supplemental linking agent isstreptavidin or avidin; (b) the linking agent is streptavidin or avidinand the supplemental linking agent is biotin; (c) the linking agent is apeptide and the supplemental linking agent is an anti-peptide antibody;or (d) the linking agent is an anti-peptide antibody and thesupplemental linking agent is a peptide.

Alternatively or additionally, in embodiments (1) and (1)(a)-(e), thebridging agent can be included, the bridging agent can be streptavidinor avidin, and the linking agents and the supplemental linking agentscan each comprise biotin.

The first and second binding reagents referenced in embodiments (1) and(1)(a)-(e), each can comprise a receptor, ligand, antibody, hapten,antigen, epitope, mimitope, aptamer, or an intercalater capable ofbinding to said first and second analytes, respectively. For example,the first and second binding reagents are antigens. Alternatively oradditionally, the first and second binding reagents are antibodies.

In embodiments (1) and (1)(a)-(e), the method can include conducting asandwich binding assay.

In one specific example of embodiments (1) and (1)(a)-(e), thecomponents combined in the combining step further comprise a firstdetection reagent that binds the first analyte and a second detectionreagent that binds the second analyte, and the first and secondcomplexes formed in the second step further comprise the first andsecond detection reagents, respectively. For example, the first andsecond detection reagents each comprise a detectable label. In anotherexample of this embodiment, the first and second binding reagents andthe first and second detection reagents are antibodies. Moreover, thefirst and second detection reagents can bind to either the first orsecond analyte, and optionally, the first and second detection reagentseach comprise a detectable label. In a specific example, the first andsecond binding reagents are antigens, the analytes are antibodiesagainst the antigens and the detection reagents compriseanti-immunoglobulin antibodies, protein A, protein G or protein L.

The method of embodiments (1) and (1)(a)-(e), can include conducting acompetitive binding assay.

Still further, the components combined in embodiments (1) and(1)(a)-(e), can further comprise a first detection reagent that competeswith the first analyte for binding to the first binding reagent and asecond detection reagent that competes with the second analyte forbinding to the second binding reagent.

In embodiments (1) and (1)(a)-(e), one or more of the followingadditional elements of the method are included: the first and seconddetection reagents can comprise detectable labels; the first and secondbinding reagents are antibodies and the first and second detectionreagents are structural analogs of the analytes; the measuring step cancomprise measuring the presence of the first and second detectablelabels via optical absorbance, fluorescence, phosphorescence,chemiluminescence, electrochemiluminescence, light scattering, ormagnetism; the first and second detectable label is anelectrochemiluminescent label and the measuring step further comprisesmeasuring an electrochemiluminescent signal and correlating the signalwith an amount of first and second analyte in the sample; the first andsecond binding domains are positioned on an electrode and the measuringstep further comprises applying a voltage waveform to the electrode togenerate electrochemiluminescence; each of the first and second bindingdomains is an element of an array of binding domains, and optionally,the array is located within a well of a multi-well plate; each of thefirst and second binding domains are each positioned on a surface of oneor more microparticles, and optionally, the particles are coded to allowfor identification of specific particles and discrimination between thefirst and second binding domains.

Embodiment (1)(f): includes the elements of embodiment (1) and one ormore additional features of embodiment (1)(a)-(e), wherein the firsttargeting agent and first targeting agent complement and the secondtargeting agent and second targeting agent complement each comprise acomplementary oligonucleotide pair. In this specific example, the firstand second binding reagents can each be antibodies. Moreover, in thisspecific example, the complementary oligonucleotide pair positioned oneach of the first and second binding domains is different and selectedfrom:

pair # Sequence (5′-3′) 1 Acatcggtagtt (SEQ ID NO: 1)Aactaccgatgt (SEQ ID NO: 2) 2 acgtcccagttg (SEQ ID NO: 3)caactgggacgt (SEQ ID NO: 4) 3 agaagaagatcc (SEQ ID NO: 5)ggatcttcttct (SEQ ID NO: 6) 4 aggttcagtgca (SEQ ID NO: 7)tgcactgaacct (SEQ ID NO: 8) 5 atcaggatacgc (SEQ ID NO: 9)gcgtatcctgat (SEQ ID NO: 10) 6 atcattaccacc (SEQ ID NO: 11)ggtggtaatgat (SEQ ID NO: 12) 7 attaacgggagc (SEQ ID NO: 13)gctcccgttaat (SEQ ID NO: 14) 8 cagaggtcttaa (SEQ ID NO: 15)ttaagacctctg (SEQ ID NO: 16) 9 caggtgtccatt (SEQ ID NO: 17)aatggacacctg (SEQ ID NO: 18) 10 catccaatccag (SEQ ID NO: 19)ctggattggatg (SEQ ID NO: 20) 11 cctacgatatac (SEQ ID NO: 21)gtatatcgtagg (SEQ ID NO: 22) 12 cgaatgtagagt (SEQ ID NO: 23)actctacattcg (SEQ ID NO: 24) 13 cggtttgagata (SEQ ID NO: 25)tatctcaaaccg (SEQ ID NO: 26) 14 cttacaacgcca (SEQ ID NO: 27)tggcgttgtaag (SEQ ID NO: 28) 15 ctttctcggcac (SEQ ID NO: 29)gtgccgagaaag (SEQ ID NO: 30) 16 gacataaagcga (SEQ ID NO: 31)tcgctttatgtc (SEQ ID NO: 32) 17 gccatagtctct (SEQ ID NO: 33)agagactatggc (SEQ ID NO: 34) 18 gctaattcacca (SEQ ID NO: 35)tggtgaattagc (SEQ ID NO: 36) 19 ggtcgtgtttca (SEQ ID NO: 37)tgaaacacgacc (SEQ ID NO: 38) 20 gttgattctgtc (SEQ ID NO: 39)gacagaatcaac (SEQ ID NO: 40) 21 tacccggaataa (SEQ ID NO: 41)ttattccgggta (SEQ ID NO: 42) 22 tgcttgacttgg (SEQ ID NO: 43)ccaagtcaagca (SEQ ID NO: 44) 23 ttccacttaggg (SEQ ID NO: 45)ccctaagtggaa (SEQ ID NO: 46) 24 ttgtctagcggc (SEQ ID NO: 47)gccgctagacaa (SEQ ID NO: 48) 25 tttcccttgcta (SEQ ID NO: 49)tagcaagggaaa (SEQ ID NO: 50)

Embodiment (1)(g): includes the elements of embodiment (1) and one ormore additional features of embodiment (1)(a)-(e), wherein the firsttargeting agent and first targeting agent complement and the secondtargeting agent and second targeting agent complement each comprise acomplementary oligonucleotide pair. In this specific example, the firstand second binding reagents can each be antibodies. Moreover, in thisspecific example, the complementary oligonucleotide pair positioned oneach of the first and second binding domains is different and selectedfrom:

Pair Sequence 1 acatcggtagtt (SEQ ID NO: 1) aactaccgatgt (SEQ ID NO: 2)3 agaagaagatcc (SEQ ID NO: 5) ggatcttcttct (SEQ ID NO: 6) 6atcattaccacc (SEQ ID NO: 11) ggtggtaatgat (SEQ ID NO: 12) 7attaacgggagc (SEQ ID NO: 13) gctcccgttaat (SEQ ID NO: 14) 8cagaggtcttaa (SEQ ID NO: 15) ttaagacctctg (SEQ ID NO: 16) 9caggtgtccatt (SEQ ID NO: 17) aatggacacctg (SEQ ID NO: 18) 11cctacgatatac (SEQ ID NO: 21) gtatatcgtagg (SEQ ID NO: 22) 12cgaatgtagagt (SEQ ID NO: 23) actctacattcg (SEQ ID NO: 24) 13cggtttgagata (SEQ ID NO: 25) tatctcaaaccg (SEQ ID NO: 26) 16gacataaagcga (SEQ ID NO: 31) tcgctttatgtc (SEQ ID NO: 32) 17gccatagtctct (SEQ ID NO: 33) agagactatggc (SEQ ID NO: 34) 18gctaattcacca (SEQ ID NO: 35) tggtgaattagc (SEQ ID NO: 36) 20gttgattctgtc (SEQ ID NO: 39) gacagaatcaac (SEQ ID NO: 40) 23ttccacttaggg (SEQ ID NO: 45) ccctaagtggaa (SEQ ID NO: 46) 25tttcccttgcta (SEQ ID NO: 49) tagcaagggaaa (SEQ ID NO: 50)

In embodiments (1)(f) and/or (1)(g), there are at least 7 bindingdomains, or at least 10 binding domains; or at least 16 binding domains;or at least 25 binding domains.

In embodiments (1) and (1)(a), each of the first and secondcomplementary targeting agents can selectively bind to one of the firstand second binding domains, respectively. For example, thecross-reactivity for binding of the complementary targeting agent toother binding domains is less than 5% of the binding to the one of thebinding domains; or the cross-reactivity for binding of each first andsecond complementary targeting agent to a third binding domain is lessthan 1% of the binding to the one of the binding domains; or thecross-reactivity for binding of each first and second complementarytargeting agent to a third binding domain is less than 0.5% of thebinding to the one of the binding domains; or the cross-reactivity forbinding of each first and second complementary targeting agent to athird binding domain is less than 0.1% of the binding to the one of thebinding domains.

In embodiments (1) and (1)(a), the first and second binding reagents caneach bind different analytes of interest; or the first and secondbinding reagents are each preferentially selective for a differentanalyte of interest.

The first and second binding reagents of any one of the foregoingembodiments can differ in the affinity and/or selectivity for differentanalytes of interest, e.g., the cross-reactivity of the first analyte tothe second binding reagent is less than 5% of the binding to the firstbinding reagent, or the cross-reactivity of the first analyte to thesecond binding reagent is less than 1% of the binding to the firstbinding reagent, or the cross-reactivity of the first analyte to thesecond binding reagent is less than 0.5% of the binding to the firstbinding reagent, or the cross-reactivity of the first analyte to thesecond binding reagent is less than 0.1% of the binding to the firstbinding reagent, or the observed cross-reactivity of an analyte ofinterest for non-specific binding reagents is less than 5% of thebinding to the binding reagent selected for binding to that analyte, orthe observed cross-reactivity of an analyte of interest for non-specificbinding reagents is less than 1% of the binding to the binding reagentselected for binding to that analyte, or the observed cross-reactivityof an analyte of interest for non-specific binding reagents is less than0.5% of the binding to the binding reagent selected for binding to thatanalyte, or the observed cross-reactivity of an analyte of interest fornon-specific binding reagents is less than 0.1% of the binding to thebinding reagent selected for binding to that analyte. Still further inthis specific example of the foregoing embodiments, the cross-reactivityof the first analyte to a binding reagent located in the second bindingdomain is less than 5% of the binding to a binding reagent located inthe first binding domain, or the cross-reactivity of the first analyteto a binding reagent located in the second binding domain is less than1% of the binding to a binding reagent located in the first bindingdomain, or the cross-reactivity of the first analyte to a bindingreagent located in the second binding domain is less than 0.5% of thebinding to a binding reagent located in the first binding domain, or thecross-reactivity of the first analyte to a binding reagent located inthe second binding domain is less than 0.1% of the binding to a bindingreagent located in the first binding domain, or the observedcross-reactivity of an analyte of interest for binding reagents locatedin a non-specific binding domain is less than 5% of the binding to thebinding reagents in the binding domain assigned to that analyte, or theobserved cross-reactivity of an analyte of interest for binding reagentslocated in a non-specific binding domain is less than 1% of the bindingto the binding reagents in the binding domain assigned to that analyte,or the observed cross-reactivity of an analyte of interest for bindingreagents located in a non-specific binding domain is less than 0.5% ofthe binding to the binding reagents in the binding domain assigned tothat analyte, or the observed cross-reactivity of an analyte of interestfor binding reagents located in a non-specific binding domain is lessthan 0.1% of the binding to the binding reagents in the binding domainassigned to that analyte.

In embodiments (1) and (1)(a), the first and second targeting agents andfirst and second targeting agent complements, respectively, can be usedto map a set of binding reagents to a set of binding domains and each ofthe binding reagents in the set bind to a different analyte of interest.For example, each of the binding reagents in the set is preferentiallyselective for a different analyte of interest, or each of the bindingreagents in the set differ in the affinity and/or selectivity fordifferent analytes of interest.

Embodiment (2): an oligonucleotide selected from:

(SEQ ID NO: 1) Acatcggtagtt (SEQ ID NO: 2) Aactaccgatgt (SEQ ID NO: 3)acgtcccagttg (SEQ ID NO: 4) caactgggacgt (SEQ ID NO: 5) agaagaagatcc(SEQ ID NO: 6) ggatcttcttct (SEQ ID NO: 7) aggttcagtgca (SEQ ID NO: 8)tgcactgaacct (SEQ ID NO: 9) atcaggatacgc (SEQ ID NO: 10) gcgtatcctgat(SEQ ID NO: 11) atcattaccacc (SEQ ID NO: 12) ggtggtaatgat(SEQ ID NO: 13) attaacgggagc (SEQ ID NO: 14) gctcccgttaat(SEQ ID NO: 15) cagaggtcttaa (SEQ ID NO: 16) ttaagacctctg(SEQ ID NO: 17) caggtgtccatt (SEQ ID NO: 18) aatggacacctg(SEQ ID NO: 19) catccaatccag (SEQ ID NO: 20) ctggattggatg(SEQ ID NO: 21) cctacgatatac (SEQ ID NO: 22) gtatatcgtagg(SEQ ID NO: 23) cgaatgtagagt (SEQ ID NO: 24) actctacattcg(SEQ ID NO: 25) cggtttgagata (SEQ ID NO: 26) tatctcaaaccg(SEQ ID NO: 27) cttacaacgcca (SEQ ID NO: 28) tggcgttgtaag(SEQ ID NO: 29) ctttctcggcac (SEQ ID NO: 30) gtgccgagaaag(SEQ ID NO: 31) gacataaagcga (SEQ ID NO: 32) tcgctttatgtc(SEQ ID NO: 33) gccatagtctct (SEQ ID NO: 34) agagactatggc(SEQ ID NO: 35) gctaattcacca (SEQ ID NO: 36) tggtgaattagc(SEQ ID NO: 37) ggtcgtgtttca (SEQ ID NO: 38) tgaaacacgacc(SEQ ID NO: 39) gttgattctgtc (SEQ ID NO: 40) gacagaatcaac(SEQ ID NO: 41) tacccggaataa (SEQ ID NO: 42) ttattccgggta(SEQ ID NO: 43) tgcttgacttgg (SEQ ID NO: 44) ccaagtcaagca(SEQ ID NO: 45) ttccacttaggg (SEQ ID NO: 46) ccctaagtggaa(SEQ ID NO: 47) ttgtctagcggc (SEQ ID NO: 48) gccgctagacaa(SEQ ID NO: 49) tttcccttgcta (SEQ ID NO: 50) tagcaagggaaa

Embodiment (3): an oligonucleotide selected from:

Pair Sequence 1 acatcggtagtt (SEQ ID NO: 1) aactaccgatgt (SEQ ID NO: 2)3 agaagaagatcc (SEQ ID NO: 5) ggatcttcttct (SEQ ID NO: 6) 6atcattaccacc (SEQ ID NO: 11) ggtggtaatgat (SEQ ID NO: 12) 7attaacgggagc (SEQ ID NO: 13) gctcccgttaat (SEQ ID NO: 14) 8cagaggtcttaa (SEQ ID NO: 15) ttaagacctctg (SEQ ID NO: 16) 9caggtgtccatt (SEQ ID NO: 17) aatggacacctg (SEQ ID NO: 18) 11cctacgatatac (SEQ ID NO: 21) gtatatcgtagg (SEQ ID NO: 22) 12cgaatgtagagt (SEQ ID NO: 23) actctacattcg (SEQ ID NO: 24) 13cggtttgagata (SEQ ID NO: 25) tatctcaaaccg (SEQ ID NO: 26) 16gacataaagcga (SEQ ID NO: 31) tcgctttatgtc (SEQ ID NO: 32) 17gccatagtctct (SEQ ID NO: 33) agagactatggc (SEQ ID NO: 34) 18gctaattcacca (SEQ ID NO: 35) tggtgaattagc (SEQ ID NO: 36) 20gttgattctgtc (SEQ ID NO: 39) gacagaatcaac (SEQ ID NO: 40) 23ttccacttaggg (SEQ ID NO: 45) ccctaagtggaa (SEQ ID NO: 46) 25tttcccttgcta (SEQ ID NO: 49) tagcaagggaaa (SEQ ID NO: 50)

Embodiment (4): a kit comprising, in separate vials, at least 4oligonucleotides, each comprising a different sequence selected from:

(SEQ ID NO: 1) Acatcggtagtt (SEQ ID NO: 2) Aactaccgatgt (SEQ ID NO: 3)acgtcccagttg (SEQ ID NO: 4) caactgggacgt (SEQ ID NO: 5) agaagaagatcc(SEQ ID NO: 6) ggatcttcttct (SEQ ID NO: 7) aggttcagtgca (SEQ ID NO: 8)tgcactgaacct (SEQ ID NO: 9) atcaggatacgc (SEQ ID NO: 10) gcgtatcctgat(SEQ ID NO: 11) atcattaccacc (SEQ ID NO: 12) ggtggtaatgat(SEQ ID NO: 13) attaacgggagc (SEQ ID NO: 14) gctcccgttaat(SEQ ID NO: 15) cagaggtcttaa (SEQ ID NO: 16) ttaagacctctg(SEQ ID NO: 17) caggtgtccatt (SEQ ID NO: 18) aatggacacctg(SEQ ID NO: 19) catccaatccag (SEQ ID NO: 20) ctggattggatg(SEQ ID NO: 21) cctacgatatac (SEQ ID NO: 22) gtatatcgtagg(SEQ ID NO: 23) cgaatgtagagt (SEQ ID NO: 24) actctacattcg(SEQ ID NO: 25) cggtttgagata (SEQ ID NO: 26) tatctcaaaccg(SEQ ID NO: 27) cttacaacgcca (SEQ ID NO: 28) tggcgttgtaag(SEQ ID NO: 29) ctttctcggcac (SEQ ID NO: 30) gtgccgagaaag(SEQ ID NO: 31) gacataaagcga (SEQ ID NO: 32) tcgctttatgtc(SEQ ID NO: 33) gccatagtctct (SEQ ID NO: 34) agagactatggc(SEQ ID NO: 35) gctaattcacca (SEQ ID NO: 36) tggtgaattagc(SEQ ID NO: 37) ggtcgtgtttca (SEQ ID NO: 38) tgaaacacgacc(SEQ ID NO: 39) gttgattctgtc (SEQ ID NO: 40) gacagaatcaac(SEQ ID NO: 41) tacccggaataa (SEQ ID NO: 42) ttattccgggta(SEQ ID NO: 43) tgcttgacttgg (SEQ ID NO: 44) ccaagtcaagca(SEQ ID NO: 45) ttccacttaggg (SEQ ID NO: 46) ccctaagtggaa(SEQ ID NO: 47) ttgtctagcggc (SEQ ID NO: 48) gccgctagacaa(SEQ ID NO: 49) tttcccttgcta (SEQ ID NO: 50) tagcaagggaaa

Embodiment (5): a kit comprising, in separate vials, at least 4oligonucleotides, each comprising a different sequence selected from:

Pair Sequence 1 acatcggtagtt (SEQ ID NO: 1) aactaccgatgt (SEQ ID NO: 2)3 agaagaagatcc (SEQ ID NO: 5) ggatcttcttct (SEQ ID NO: 6) 6atcattaccacc (SEQ ID NO: 11) ggtggtaatgat (SEQ ID NO: 12) 7attaacgggagc (SEQ ID NO: 13) gctcccgttaat (SEQ ID NO: 14) 8cagaggtcttaa (SEQ ID NO: 15) ttaagacctctg (SEQ ID NO: 16) 9caggtgtccatt (SEQ ID NO: 17) aatggacacctg (SEQ ID NO: 18) 11cctacgatatac (SEQ ID NO: 21) gtatatcgtagg (SEQ ID NO: 22) 12cgaatgtagagt (SEQ ID NO: 23) actctacattcg (SEQ ID NO: 24) 13cggtttgagata (SEQ ID NO: 25) tatctcaaaccg (SEQ ID NO: 26) 16gacataaagcga (SEQ ID NO: 31) tcgctttatgtc (SEQ ID NO: 32) 17gccatagtctct (SEQ ID NO: 33) agagactatggc (SEQ ID NO: 34) 18gctaattcacca (SEQ ID NO: 35) tggtgaattagc (SEQ ID NO: 36) 20gttgattctgtc (SEQ ID NO: 39) gacagaatcaac (SEQ ID NO: 40) 23ttccacttaggg (SEQ ID NO: 45) ccctaagtggaa (SEQ ID NO: 46) 25tttcccttgcta (SEQ ID NO: 49) tagcaagggaaa (SEQ ID NO: 50)

In the kit of embodiments (4) and (5), the kit can comprise at least 7oligonucleotides, or at least 10 oligonucleotides, or at least 16oligonucleotides, or at least 25 oligonucleotides. Moreover, each of theoligonucleotides of these specific embodiments can be modified with alinking agent comprising a biotin, streptavidin, avidin, amino group,thiol group, aldehyde group, hydrazide group, azide group, alkyne group,maleimide group or iodoacetamide group. In another specific example ofembodiments (4) and (5), each of the at least 4 oligonucleotides can becoupled to a different antibody, and optionally, each different sequenceis not a complement of an additional different sequence of the at least4 oligonucleotides.

Embodiment (6): a mixture comprising at least 4 oligonucleotides, eachcomprising a different sequence selected from:

(SEQ ID NO: 1) Acatcggtagtt (SEQ ID NO: 2) Aactaccgatgt (SEQ ID NO: 3)acgtcccagttg (SEQ ID NO: 4) caactgggacgt (SEQ ID NO: 5) agaagaagatcc(SEQ ID NO: 6) ggatcttcttct (SEQ ID NO: 7) aggttcagtgca (SEQ ID NO: 8)tgcactgaacct (SEQ ID NO: 9) atcaggatacgc (SEQ ID NO: 10) gcgtatcctgat(SEQ ID NO: 11) atcattaccacc (SEQ ID NO: 12) ggtggtaatgat(SEQ ID NO: 13) attaacgggagc (SEQ ID NO: 14) gctcccgttaat(SEQ ID NO: 15) cagaggtcttaa (SEQ ID NO: 16) ttaagacctctg(SEQ ID NO: 17) caggtgtccatt (SEQ ID NO: 18) aatggacacctg(SEQ ID NO: 19) catccaatccag (SEQ ID NO: 20) ctggattggatg(SEQ ID NO: 21) cctacgatatac (SEQ ID NO: 22) gtatatcgtagg(SEQ ID NO: 23) cgaatgtagagt (SEQ ID NO: 24) actctacattcg(SEQ ID NO: 25) cggtttgagata (SEQ ID NO: 26) tatctcaaaccg(SEQ ID NO: 27) cttacaacgcca (SEQ ID NO: 28) tggcgttgtaag(SEQ ID NO: 29) ctttctcggcac (SEQ ID NO: 30) gtgccgagaaag(SEQ ID NO: 31) gacataaagcga (SEQ ID NO: 32) tcgctttatgtc(SEQ ID NO: 33) gccatagtctct (SEQ ID NO: 34) agagactatggc(SEQ ID NO: 35) gctaattcacca (SEQ ID NO: 36) tggtgaattagc(SEQ ID NO: 37) ggtcgtgtttca (SEQ ID NO: 38) tgaaacacgacc(SEQ ID NO: 39) gttgattctgtc (SEQ ID NO: 40) gacagaatcaac(SEQ ID NO: 41) tacccggaataa (SEQ ID NO: 42) ttattccgggta(SEQ ID NO: 43) tgcttgacttgg (SEQ ID NO: 44) ccaagtcaagca(SEQ ID NO: 45) ttccacttaggg (SEQ ID NO: 46) ccctaagtggaa(SEQ ID NO: 47) ttgtctagcggc (SEQ ID NO: 48) gccgctagacaa(SEQ ID NO: 49) tttcccttgcta (SEQ ID NO: 50) tagcaagggaaa

wherein each of the different sequences are coupled to a differentantibody.

Embodiment (7): a mixture comprising at least 4 oligonucleotides, eachcomprising a different sequence selected from:

Pair Sequence 1 acatcggtagtt (SEQ ID NO: 1) aactaccgatgt (SEQ ID NO: 2)3 agaagaagatcc (SEQ ID NO: 5) ggatcttcttct (SEQ ID NO: 6) 6atcattaccacc (SEQ ID NO: 11) ggtggtaatgat (SEQ ID NO: 12) 7attaacgggagc (SEQ ID NO: 13) gctcccgttaat (SEQ ID NO: 14) 8cagaggtcttaa (SEQ ID NO: 15) ttaagacctctg (SEQ ID NO: 16) 9caggtgtccatt (SEQ ID NO: 17) aatggacacctg (SEQ ID NO: 18) 11cctacgatatac (SEQ ID NO: 21) gtatatcgtagg (SEQ ID NO: 22) 12cgaatgtagagt (SEQ ID NO: 23) actctacattcg (SEQ ID NO: 24) 13cggtttgagata (SEQ ID NO: 25) tatctcaaaccg (SEQ ID NO: 26) 16gacataaagcga (SEQ ID NO: 31) tcgctttatgtc (SEQ ID NO: 32) 17gccatagtctct (SEQ ID NO: 33) agagactatggc (SEQ ID NO: 34) 18gctaattcacca (SEQ ID NO: 35) tggtgaattagc (SEQ ID NO: 36) 20gttgattctgtc (SEQ ID NO: 39) gacagaatcaac (SEQ ID NO: 40) 23ttccacttaggg (SEQ ID NO: 45) ccctaagtggaa (SEQ ID NO: 46) 25tttcccttgcta (SEQ ID NO: 49) tagcaagggaaa (SEQ ID NO: 50)

wherein each of the different sequences are coupled to a differentantibody.

In embodiments (6) and (7), the mixture can include at least 7oligonucleotides, or at least 10 oligonucleotides; or at least 16oligonucleotides; or at least 25 oligonucleotides. Moreover, in aspecific example of embodiments (6) and (7), each different sequence isnot a complement of an additional different sequence of the at least 4oligonucleotides.

Embodiment (8): an array comprising a plurality of at least 4 bindingdomains, wherein one or more, and optionally, each binding domain hasimmobilized thereon a different oligonucleotide sequence selected from:

(SEQ ID NO: 1) Acatcggtagtt (SEQ ID NO: 2) Aactaccgatgt (SEQ ID NO: 3)acgtcccagttg (SEQ ID NO: 4) caactgggacgt (SEQ ID NO: 5) agaagaagatcc(SEQ ID NO: 6) ggatcttcttct (SEQ ID NO: 7) aggttcagtgca (SEQ ID NO: 8)tgcactgaacct (SEQ ID NO: 9) atcaggatacgc (SEQ ID NO: 10) gcgtatcctgat(SEQ ID NO: 11) atcattaccacc (SEQ ID NO: 12) ggtggtaatgat(SEQ ID NO: 13) attaacgggagc (SEQ ID NO: 14) gctcccgttaat(SEQ ID NO: 15) cagaggtcttaa (SEQ ID NO: 16) ttaagacctctg(SEQ ID NO: 17) caggtgtccatt (SEQ ID NO: 18) aatggacacctg(SEQ ID NO: 19) catccaatccag (SEQ ID NO: 20) ctggattggatg(SEQ ID NO: 21) cctacgatatac (SEQ ID NO: 22) gtatatcgtagg(SEQ ID NO: 23) cgaatgtagagt (SEQ ID NO: 24) actctacattcg(SEQ ID NO: 25) cggtttgagata (SEQ ID NO: 26) tatctcaaaccg(SEQ ID NO: 27) cttacaacgcca (SEQ ID NO: 28) tggcgttgtaag(SEQ ID NO: 29) ctttctcggcac (SEQ ID NO: 30) gtgccgagaaag(SEQ ID NO: 31) gacataaagcga (SEQ ID NO: 32) tcgctttatgtc(SEQ ID NO: 33) gccatagtctct (SEQ ID NO: 34) agagactatggc(SEQ ID NO: 35) gctaattcacca (SEQ ID NO: 36) tggtgaattagc(SEQ ID NO: 37) ggtcgtgtttca (SEQ ID NO: 38) tgaaacacgacc(SEQ ID NO: 39) gttgattctgtc (SEQ ID NO: 40) gacagaatcaac(SEQ ID NO: 41) tacccggaataa (SEQ ID NO: 42) ttattccgggta(SEQ ID NO: 43) tgcttgacttgg (SEQ ID NO: 44) ccaagtcaagca(SEQ ID NO: 45) ttccacttaggg (SEQ ID NO: 46) ccctaagtggaa(SEQ ID NO: 47) ttgtctagcggc (SEQ ID NO: 48) gccgctagacaa(SEQ ID NO: 49) tttcccttgcta (SEQ ID NO: 50) tagcaagggaaa

Embodiment (9): an array comprising a plurality of at least 4 bindingdomains, wherein one or more and optionally, each binding domain hasimmobilized thereon a different oligonucleotide sequence selected from:

Pair Sequence 1 acatcggtagtt (SEQ ID NO: 1) aactaccgatgt (SEQ ID NO: 2)3 agaagaagatcc (SEQ ID NO: 5) ggatcttcttct (SEQ ID NO: 6) 6atcattaccacc (SEQ ID NO: 11) ggtggtaatgat (SEQ ID NO: 12) 7attaacgggagc (SEQ ID NO: 13) gctcccgttaat (SEQ ID NO: 14) 8cagaggtcttaa (SEQ ID NO: 15) ttaagacctctg (SEQ ID NO: 16) 9caggtgtccatt (SEQ ID NO: 17) aatggacacctg (SEQ ID NO: 18) 11cctacgatatac (SEQ ID NO: 21) gtatatcgtagg (SEQ ID NO: 22) 12cgaatgtagagt (SEQ ID NO: 23) actctacattcg (SEQ ID NO: 24) 13cggtttgagata (SEQ ID NO: 25) tatctcaaaccg (SEQ ID NO: 26) 16gacataaagcga (SEQ ID NO: 31) tcgctttatgtc (SEQ ID NO: 32) 17gccatagtctct (SEQ ID NO: 33) agagactatggc (SEQ ID NO: 34) 18gctaattcacca (SEQ ID NO: 35) tggtgaattagc (SEQ ID NO: 36) 20gttgattctgtc (SEQ ID NO: 39) gacagaatcaac (SEQ ID NO: 40) 23ttccacttaggg (SEQ ID NO: 45) ccctaagtggaa (SEQ ID NO: 46) 25tttcccttgcta (SEQ ID NO: 49) tagcaagggaaa (SEQ ID NO: 50)

In embodiments (8) and (9), the array can comprise at least 7 bindingdomains, or at least 10 binding domains, or at least 16 binding domains,or at least 25 binding domains, and optionally, each different sequenceis not a complement of an additional different sequence of the at least4 oligonucleotides

Embodiment (10): a multi-well plate having one or more copies of anoligonucleotide array within at least one well(s) of the plate, thearray is positioned on a plurality of binding domains, wherein one ormore and optionally, at least 4 of the binding domains have immobilizedthereon a different oligonucleotide sequence selected from:

(SEQ ID NO: 1) Acatcggtagtt (SEQ ID NO: 2) Aactaccgatgt (SEQ ID NO: 3)acgtcccagttg (SEQ ID NO: 4) caactgggacgt (SEQ ID NO: 5) agaagaagatcc(SEQ ID NO: 6) ggatcttcttct (SEQ ID NO: 7) aggttcagtgca (SEQ ID NO: 8)tgcactgaacct (SEQ ID NO: 9) atcaggatacgc (SEQ ID NO: 10) gcgtatcctgat(SEQ ID NO: 11) atcattaccacc (SEQ ID NO: 12) ggtggtaatgat(SEQ ID NO: 13) attaacgggagc (SEQ ID NO: 14) gctcccgttaat(SEQ ID NO: 15) cagaggtcttaa (SEQ ID NO: 16) ttaagacctctg(SEQ ID NO: 17) caggtgtccatt (SEQ ID NO: 18) aatggacacctg(SEQ ID NO: 19) catccaatccag (SEQ ID NO: 20) ctggattggatg(SEQ ID NO: 21) cctacgatatac (SEQ ID NO: 22) gtatatcgtagg(SEQ ID NO: 23) cgaatgtagagt (SEQ ID NO: 24) actctacattcg(SEQ ID NO: 25) cggtttgagata (SEQ ID NO: 26) tatctcaaaccg(SEQ ID NO: 27) cttacaacgcca (SEQ ID NO: 28) tggcgttgtaag(SEQ ID NO: 29) ctttctcggcac (SEQ ID NO: 30) gtgccgagaaag(SEQ ID NO: 31) gacataaagcga (SEQ ID NO: 32) tcgctttatgtc(SEQ ID NO: 33) gccatagtctct (SEQ ID NO: 34) agagactatggc(SEQ ID NO: 35) gctaattcacca (SEQ ID NO: 36) tggtgaattagc(SEQ ID NO: 37) ggtcgtgtttca (SEQ ID NO: 38) tgaaacacgacc(SEQ ID NO: 39) gttgattctgtc (SEQ ID NO: 40) gacagaatcaac(SEQ ID NO: 41) tacccggaataa (SEQ ID NO: 42) ttattccgggta(SEQ ID NO: 43) tgcttgacttgg (SEQ ID NO: 44) ccaagtcaagca(SEQ ID NO: 45) ttccacttaggg (SEQ ID NO: 46) ccctaagtggaa(SEQ ID NO: 47) ttgtctagcggc (SEQ ID NO: 48) gccgctagacaa(SEQ ID NO: 49) tttcccttgcta (SEQ ID NO: 50) tagcaagggaaa

Embodiment (11): a multi-well plate having one or more copies of anoligonucleotide array within at least one well(s) of the plate, thearray is positioned on a plurality of binding domains, wherein one ormore and optionally, at least 4 of the binding domains have immobilizedthereon a different oligonucleotide sequence selected from:

Pair Sequence 1 acatcggtagtt (SEQ ID NO: 1) aactaccgatgt (SEQ ID NO: 2)3 agaagaagatcc (SEQ ID NO: 5) ggatcttcttct (SEQ ID NO: 6) 6atcattaccacc (SEQ ID NO: 11) ggtggtaatgat (SEQ ID NO: 12) 7attaacgggagc (SEQ ID NO: 13) gctcccgttaat (SEQ ID NO: 14) 8cagaggtcttaa (SEQ ID NO: 15) ttaagacctctg (SEQ ID NO: 16) 9caggtgtccatt (SEQ ID NO: 17) aatggacacctg (SEQ ID NO: 18) 11cctacgatatac (SEQ ID NO: 21) gtatatcgtagg (SEQ ID NO: 22) 12cgaatgtagagt (SEQ ID NO: 23) actctacattcg (SEQ ID NO: 24) 13cggtttgagata (SEQ ID NO: 25) tatctcaaaccg (SEQ ID NO: 26) 16gacataaagcga (SEQ ID NO: 31) tcgctttatgtc (SEQ ID NO: 32) 17gccatagtctct (SEQ ID NO: 33) agagactatggc (SEQ ID NO: 34) 18gctaattcacca (SEQ ID NO: 35) tggtgaattagc (SEQ ID NO: 36) 20gttgattctgtc (SEQ ID NO: 39) gacagaatcaac (SEQ ID NO: 40) 23ttccacttaggg (SEQ ID NO: 45) ccctaagtggaa (SEQ ID NO: 46) 25tttcccttgcta (SEQ ID NO: 49) tagcaagggaaa (SEQ ID NO: 50)

The plate of embodiments (10) and (11) can include at least 24 wells, orat least 96 wells, or at least 384 wells, and/or optionally, the arraycomprises at least 7 oligonucleotides, or at least 10 oligonucleotides,or at least 16 oligonucleotides, or at least 25 oligonucleotides,wherein optionally, each different sequence is not a complement of anadditional different sequence of the at least 4 oligonucleotides

Embodiment (12): a kit comprising a set of at least 4 microparticlereagents, in one or more vials, wherein each microparticle reagent ofthe set comprises a microparticle having immobilized thereon a differentoligonucleotide sequence selected from:

(SEQ ID NO: 1) Acatcggtagtt (SEQ ID NO: 2) Aactaccgatgt (SEQ ID NO: 3)acgtcccagttg (SEQ ID NO: 4) caactgggacgt (SEQ ID NO: 5) agaagaagatcc(SEQ ID NO: 6) ggatcttcttct (SEQ ID NO: 7) aggttcagtgca (SEQ ID NO: 8)tgcactgaacct (SEQ ID NO: 9) atcaggatacgc (SEQ ID NO: 10) gcgtatcctgat(SEQ ID NO: 11) atcattaccacc (SEQ ID NO: 12) ggtggtaatgat(SEQ ID NO: 13) attaacgggagc (SEQ ID NO: 14) gctcccgttaat(SEQ ID NO: 15) cagaggtcttaa (SEQ ID NO: 16) ttaagacctctg(SEQ ID NO: 17) caggtgtccatt (SEQ ID NO: 18) aatggacacctg(SEQ ID NO: 19) catccaatccag (SEQ ID NO: 20) ctggattggatg(SEQ ID NO: 21) cctacgatatac (SEQ ID NO: 22) gtatatcgtagg(SEQ ID NO: 23) cgaatgtagagt (SEQ ID NO: 24) actctacattcg(SEQ ID NO: 25) cggtttgagata (SEQ ID NO: 26) tatctcaaaccg(SEQ ID NO: 27) cttacaacgcca (SEQ ID NO: 28) tggcgttgtaag(SEQ ID NO: 29) ctttctcggcac (SEQ ID NO: 30) gtgccgagaaag(SEQ ID NO: 31) gacataaagcga (SEQ ID NO: 32) tcgctttatgtc(SEQ ID NO: 33) gccatagtctct (SEQ ID NO: 34) agagactatggc(SEQ ID NO: 35) gctaattcacca (SEQ ID NO: 36) tggtgaattagc(SEQ ID NO: 37) ggtcgtgtttca (SEQ ID NO: 38) tgaaacacgacc(SEQ ID NO: 39) gttgattctgtc (SEQ ID NO: 40) gacagaatcaac(SEQ ID NO: 41) tacccggaataa (SEQ ID NO: 42) ttattccgggta(SEQ ID NO: 43) tgcttgacttgg (SEQ ID NO: 44) ccaagtcaagca(SEQ ID NO: 45) ttccacttaggg (SEQ ID NO: 46) ccctaagtggaa(SEQ ID NO: 47) ttgtctagcggc (SEQ ID NO: 48) gccgctagacaa(SEQ ID NO: 49) tttcccttgcta (SEQ ID NO: 50) tagcaagggaaa

Embodiment (13): a kit comprising a set of at least 4 microparticlereagents, in one or more vials, wherein each microparticle reagent ofthe set comprises a microparticle having immobilized thereon a differentoligonucleotide sequence selected from:

Pair Sequence 1 acatcggtagtt (SEQ ID NO: 1) aactaccgatgt (SEQ ID NO: 2)3 agaagaagatcc (SEQ ID NO: 5) ggatcttcttct (SEQ ID NO: 6) 6atcattaccacc (SEQ ID NO: 11) ggtggtaatgat (SEQ ID NO: 12) 7attaacgggagc (SEQ ID NO: 13) gctcccgttaat (SEQ ID NO: 14) 8cagaggtcttaa (SEQ ID NO: 15) ttaagacctctg (SEQ ID NO: 16) 9caggtgtccatt (SEQ ID NO: 17) aatggacacctg (SEQ ID NO: 18) 11cctacgatatac (SEQ ID NO: 21) gtatatcgtagg (SEQ ID NO: 22) 12cgaatgtagagt (SEQ ID NO: 23) actctacattcg (SEQ ID NO: 24) 13cggtttgagata (SEQ ID NO: 25) tatctcaaaccg (SEQ ID NO: 26) 16gacataaagcga (SEQ ID NO: 31) tcgctttatgtc (SEQ ID NO: 32) 17gccatagtctct (SEQ ID NO: 33) agagactatggc (SEQ ID NO: 34) 18gctaattcacca (SEQ ID NO: 35) tggtgaattagc (SEQ ID NO: 36) 20gttgattctgtc (SEQ ID NO: 39) gacagaatcaac (SEQ ID NO: 40) 23ttccacttaggg (SEQ ID NO: 45) ccctaagtggaa (SEQ ID NO: 46) 25tttcccttgcta (SEQ ID NO: 49) tagcaagggaaa (SEQ ID NO: 50)

The kit of embodiments (12) and (13) can include a set of at least 7microparticles, or a set of at least 10 microparticles, or a set of atleast 16 microparticles, or a set of at least 25 microparticles, and/oroptionally, each different sequence is not a complement of an additionaldifferent sequence of the at least 4 oligonucleotides.

The microparticles of embodiments (12) and (13) can be coded and thedifferent microparticle reagents can have different identifying codes.

Embodiment (14): a kit comprising a multi-well plate having one or morecopies of an oligonucleotide array within at least one well(s) of theplate, the array is positioned on a plurality of binding domains,wherein one or more and optionally, at least 4 of the binding domainshave immobilized thereon a different oligonucleotide sequence selectedfrom:

(SEQ ID NO: 1) Acatcggtagtt (SEQ ID NO: 2) Aactaccgatgt (SEQ ID NO: 3)acgtcccagttg (SEQ ID NO: 4) caactgggacgt (SEQ ID NO: 5) agaagaagatcc(SEQ ID NO: 6) ggatcttcttct (SEQ ID NO: 7) aggttcagtgca (SEQ ID NO: 8)tgcactgaacct (SEQ ID NO: 9) atcaggatacgc (SEQ ID NO: 10) gcgtatcctgat(SEQ ID NO: 11) atcattaccacc (SEQ ID NO: 12) ggtggtaatgat(SEQ ID NO: 13) attaacgggagc (SEQ ID NO: 14) gctcccgttaat(SEQ ID NO: 15) cagaggtcttaa (SEQ ID NO: 16) ttaagacctctg(SEQ ID NO: 17) caggtgtccatt (SEQ ID NO: 18) aatggacacctg(SEQ ID NO: 19) catccaatccag (SEQ ID NO: 20) ctggattggatg(SEQ ID NO: 21) cctacgatatac (SEQ ID NO: 22) gtatatcgtagg(SEQ ID NO: 23) cgaatgtagagt (SEQ ID NO: 24) actctacattcg(SEQ ID NO: 25) cggtttgagata (SEQ ID NO: 26) tatctcaaaccg(SEQ ID NO: 27) cttacaacgcca (SEQ ID NO: 28) tggcgttgtaag(SEQ ID NO: 29) ctttctcggcac (SEQ ID NO: 30) gtgccgagaaag(SEQ ID NO: 31) gacataaagcga (SEQ ID NO: 32) tcgctttatgtc(SEQ ID NO: 33) gccatagtctct (SEQ ID NO: 34) agagactatggc(SEQ ID NO: 35) gctaattcacca (SEQ ID NO: 36) tggtgaattagc(SEQ ID NO: 37) ggtcgtgtttca (SEQ ID NO: 38) tgaaacacgacc(SEQ ID NO: 39) gttgattctgtc (SEQ ID NO: 40) gacagaatcaac(SEQ ID NO: 41) tacccggaataa (SEQ ID NO: 42) ttattccgggta(SEQ ID NO: 43) tgcttgacttgg (SEQ ID NO: 44) ccaagtcaagca(SEQ ID NO: 45) ttccacttaggg (SEQ ID NO: 46) ccctaagtggaa(SEQ ID NO: 47) ttgtctagcggc (SEQ ID NO: 48) gccgctagacaa(SEQ ID NO: 49) tttcccttgcta (SEQ ID NO: 50) tagcaagggaaa

and

-   -   (i) a set of oligonucleotides comprised of two or more        oligonucleotides selected from:

(SEQ ID NO: 1) Acatcggtagtt (SEQ ID NO: 2) Aactaccgatgt (SEQ ID NO: 3)acgtcccagttg (SEQ ID NO: 4) caactgggacgt (SEQ ID NO: 5) agaagaagatcc(SEQ ID NO: 6) ggatcttcttct (SEQ ID NO: 7) aggttcagtgca (SEQ ID NO: 8)tgcactgaacct (SEQ ID NO: 9) atcaggatacgc (SEQ ID NO: 10) gcgtatcctgat(SEQ ID NO: 11) atcattaccacc (SEQ ID NO: 12) ggtggtaatgat(SEQ ID NO: 13) attaacgggagc (SEQ ID NO: 14) gctcccgttaat(SEQ ID NO: 15) cagaggtcttaa (SEQ ID NO: 16) ttaagacctctg(SEQ ID NO: 17) caggtgtccatt (SEQ ID NO: 18) aatggacacctg(SEQ ID NO: 19) catccaatccag (SEQ ID NO: 20) ctggattggatg(SEQ ID NO: 21) cctacgatatac (SEQ ID NO: 22) gtatatcgtagg(SEQ ID NO: 23) cgaatgtagagt (SEQ ID NO: 24) actctacattcg(SEQ ID NO: 25) cggtttgagata (SEQ ID NO: 26) tatctcaaaccg(SEQ ID NO: 27) cttacaacgcca (SEQ ID NO: 28) tggcgttgtaag(SEQ ID NO: 29) ctttctcggcac (SEQ ID NO: 30) gtgccgagaaag(SEQ ID NO: 31) gacataaagcga (SEQ ID NO: 32) tcgctttatgtc(SEQ ID NO: 33) gccatagtctct (SEQ ID NO: 34) agagactatggc(SEQ ID NO: 35) gctaattcacca (SEQ ID NO: 36) tggtgaattagc(SEQ ID NO: 37) ggtcgtgtttca (SEQ ID NO: 38) tgaaacacgacc(SEQ ID NO: 39) gttgattctgtc (SEQ ID NO: 40) gacagaatcaac(SEQ ID NO: 41) tacccggaataa (SEQ ID NO: 42) ttattccgggta(SEQ ID NO: 43) tgcttgacttgg (SEQ ID NO: 44) ccaagtcaagca(SEQ ID NO: 45) ttccacttaggg (SEQ ID NO: 46) ccctaagtggaa(SEQ ID NO: 47) ttgtctagcggc (SEQ ID NO: 48) gccgctagacaa(SEQ ID NO: 49) tttcccttgcta (SEQ ID NO: 50) tagcaagggaaa

wherein the oligonucleotides in component (ii) are complementary to theoligonucleotides in component (i).

Embodiment (15): a kit comprising a multi-well plate having one or morecopies of an oligonucleotide array within at least one well(s) of theplate, the array is positioned on a plurality of binding domains,wherein one or more and optionally, at least 4 of the binding domainshave immobilized thereon a different oligonucleotide sequence selectedfrom:

Pair Sequence 1 acatcggtagtt (SEQ ID NO: 1) aactaccgatgt (SEQ ID NO: 2)3 agaagaagatcc (SEQ ID NO: 5) ggatcttcttct (SEQ ID NO: 6) 6atcattaccacc (SEQ ID NO: 11) ggtggtaatgat (SEQ ID NO: 12) 7attaacgggagc (SEQ ID NO: 13) gctcccgttaat (SEQ ID NO: 14) 8cagaggtcttaa (SEQ ID NO: 15) ttaagacctctg (SEQ ID NO: 16) 9caggtgtccatt (SEQ ID NO: 17) aatggacacctg (SEQ ID NO: 18) 11cctacgatatac (SEQ ID NO: 21) gtatatcgtagg (SEQ ID NO: 22) 12cgaatgtagagt (SEQ ID NO: 23) actctacattcg (SEQ ID NO: 24) 13cggtttgagata (SEQ ID NO: 25) tatctcaaaccg (SEQ ID NO: 26) 16gacataaagcga (SEQ ID NO: 31) tcgctttatgtc (SEQ ID NO: 32) 17gccatagtctct (SEQ ID NO: 33) agagactatggc (SEQ ID NO: 34) 18gctaattcacca (SEQ ID NO: 35) tggtgaattagc (SEQ ID NO: 36) 20gttgattctgtc (SEQ ID NO: 39) gacagaatcaac (SEQ ID NO: 40) 23ttccacttaggg (SEQ ID NO: 45) ccctaagtggaa (SEQ ID NO: 46) 25tttcccttgcta (SEQ ID NO: 49) tagcaagggaaa (SEQ ID NO: 50)

and

-   -   (i) a set of oligonucleotides comprised of two or more        oligonucleotides selected from:

Pair Sequence 1 acatcggtagtt (SEQ ID NO: 1) aactaccgatgt (SEQ ID NO: 2)3 agaagaagatcc (SEQ ID NO: 5) ggatcttcttct (SEQ ID NO: 6) 6atcattaccacc (SEQ ID NO: 11) ggtggtaatgat (SEQ ID NO: 12) 7attaacgggagc (SEQ ID NO: 13) gctcccgttaat (SEQ ID NO: 14) 8cagaggtcttaa (SEQ ID NO: 15) ttaagacctctg (SEQ ID NO: 16) 9caggtgtccatt (SEQ ID NO: 17) aatggacacctg (SEQ ID NO: 18) 11cctacgatatac (SEQ ID NO: 21) gtatatcgtagg (SEQ ID NO: 22) 12cgaatgtagagt (SEQ ID NO: 23) actctacattcg (SEQ ID NO: 24) 13cggtttgagata (SEQ ID NO: 25) tatctcaaaccg (SEQ ID NO: 26) 16gacataaagcga (SEQ ID NO: 31) tcgctttatgtc (SEQ ID NO: 32) 17gccatagtctct (SEQ ID NO: 33) agagactatggc (SEQ ID NO: 34) 18gctaattcacca (SEQ ID NO: 35) tggtgaattagc (SEQ ID NO: 36) 20gttgattctgtc (SEQ ID NO: 39) gacagaatcaac (SEQ ID NO: 40) 23ttccacttaggg (SEQ ID NO: 45) ccctaagtggaa (SEQ ID NO: 46) 25tttcccttgcta (SEQ ID NO: 49) tagcaagggaaa (SEQ ID NO: 50)

wherein the oligonucleotides in component (ii) are complementary to theoligonucleotides in component (i).

Embodiment (16): a method of conducting a binding assay for a pluralityof analytes comprising: (a) contacting a sample with two or more bindingdomains linked to at least a first and second binding reagent that eachbind a first and second analyte, respectively, of the plurality ofanalytes to form complexes comprising the first analyte bound to thefirst binding reagent and the second analyte bound to the second bindingreagent, wherein (x) the first binding domain comprises a first bindingreagent complex comprising (i) a first targeting agent bound to thefirst binding domain and to a first targeting agent complement; and (ii)the first binding reagent bound to the first targeting agent complementvia a linking complex; and (y) the second binding domain comprises asecond binding reagent complex comprising (i) a second targeting agentbound to the second binding domain and to a second targeting agentcomplement; and (ii) the second binding reagent bound to the secondtargeting agent complement via a linking complex; (b) contacting thefirst and second binding reagent complexes with a plurality of detectionreagents comprising a first detection reagent that binds the firstanalyte or a complex comprising the first analyte, and a seconddetection reagent that binds the second analyte or a complex comprisingthe second analyte; and (c) measuring the amount of the first and secondanalytes bound to the two or more binding domains.

In embodiment (16), one or more of the following features can beadopted: the first and second binding reagents each comprise a receptor,ligand, antibody, hapten, antigen, epitope, mimitope, aptamer, or anintercalater capable of binding to the first and second analyte,respectively, e.g., the first and second binding reagents each comprisean antibody capable of binding to the first and second analyte,respectively; the first targeting agent and the first targeting agentcomplement comprise an oligonucleotide and a complementaryoligonucleotide, a receptor-ligand pair, an antigen-antibody pair, ahapten-antibody pair, an epitope-antibody pair, an mimitope-antibodypair, an aptamer-target molecule pair, a hybridization partners, or anintercalater-target molecule pair, the second targeting agent and thesecond targeting agent complement comprise an oligonucleotide and acomplementary oligonucleotide, a receptor-ligand pair, anantigen-antibody pair, a hapten-antibody pair, an epitope-antibody pair,an mimitope-antibody pair, an aptamer-target molecule pair, ahybridization partners, or an intercalater-target molecule pair; thefirst targeting agent and the first targeting agent complement comprisean oligonucleotide and a complementary oligonucleotide, respectively;the second targeting agent and the second targeting agent complementcomprise an oligonucleotide and a complementary oligonucleotide,respectively; the linking complex comprises a linking agent and asupplemental linking agent connected thereto, e.g., the linking complexis formed by a binding interaction between (a) a thiol group and amaleimide or iodoacetamide; (b) an aldehyde and a hydrazide; or (c) analkyne and an azide; or the (a) the linking agent is biotin andsupplemental linking agent is streptavidin or avidin; (b) the linkingagent is streptavidin or avidin and the supplemental linking agent isbiotin; (c) the linking agent is a peptide and the supplemental linkingagent is an anti-peptide antibody; or (d) the linking agent is ananti-peptide antibody and the supplemental linking agent is a peptide.

Moreover, embodiment (16) can optionally include one or more of thefollowing features: each of the plurality of detection reagents comprisea detectable label; a subset of the plurality of detection reagentscomprise a detectable label; the measuring step comprises measuring thepresence of the detectable label in the sample via optical absorbance,fluorescence, phosphorescence, chemiluminescence,electrochemiluminescence, light scattering, or magnetism; the detectablelabel is an electrochemiluminescent label and the measuring stepcomprises measuring an electrochemiluminescent signal and correlatingthe signal with an amount of analyte in the sample, e.g., the two ormore binding domains are positioned on an electrode and the measuringstep further comprises applying a voltage waveform to the electrode togenerate electrochemiluminescence, and optionally, the two or morebinding domains are located within one or more wells of a multi-wellplate.

Embodiment (16) can include conducting a sandwich immunoassay or acompetitive immunoassay.

In embodiment (16), the first targeting agent and the first targetingagent complement can comprise a pair of oligonucleotides, wherein thepair is selected from:

pair # Sequence (5′-3′) 1 Acatcggtagtt (SEQ ID NO: 1)Aactaccgatgt (SEQ ID NO: 2) 2 acgtcccagttg (SEQ ID NO: 3)caactgggacgt (SEQ ID NO: 4) 3 agaagaagatcc (SEQ ID NO: 5)ggatcttcttct (SEQ ID NO: 6) 4 aggttcagtgca (SEQ ID NO: 7)tgcactgaacct (SEQ ID NO: 8) 5 atcaggatacgc (SEQ ID NO: 9)gcgtatcctgat (SEQ ID NO: 10) 6 atcattaccacc (SEQ ID NO: 11)ggtggtaatgat (SEQ ID NO: 12) 7 attaacgggagc (SEQ ID NO: 13)gctcccgttaat (SEQ ID NO: 14) 8 cagaggtcttaa (SEQ ID NO: 15)ttaagacctctg (SEQ ID NO: 16) 9 caggtgtccatt (SEQ ID NO: 17)aatggacacctg (SEQ ID NO: 18) 10 catccaatccag (SEQ ID NO: 19)ctggattggatg (SEQ ID NO: 20) 11 cctacgatatac (SEQ ID NO: 21)gtatatcgtagg (SEQ ID NO: 22) 12 cgaatgtagagt (SEQ ID NO: 23)actctacattcg (SEQ ID NO: 24) 13 cggtttgagata (SEQ ID NO: 25)tatctcaaaccg (SEQ ID NO: 26) 14 cttacaacgcca (SEQ ID NO: 27)tggcgttgtaag (SEQ ID NO: 28) 15 ctttctcggcac (SEQ ID NO: 29)gtgccgagaaag (SEQ ID NO: 30) 16 gacataaagcga (SEQ ID NO: 31)tcgctttatgtc (SEQ ID NO: 32) 17 gccatagtctct (SEQ ID NO: 33)agagactatggc (SEQ ID NO: 34) 18 gctaattcacca (SEQ ID NO: 35)tggtgaattagc (SEQ ID NO: 36) 19 ggtcgtgtttca (SEQ ID NO: 37)tgaaacacgacc (SEQ ID NO: 38) 20 gttgattctgtc (SEQ ID NO: 39)gacagaatcaac (SEQ ID NO: 40) 21 tacccggaataa (SEQ ID NO: 41)ttattccgggta (SEQ ID NO: 42) 22 tgcttgacttgg (SEQ ID NO: 43)ccaagtcaagca (SEQ ID NO: 44) 23 ttccacttaggg (SEQ ID NO: 45)ccctaagtggaa (SEQ ID NO: 46) 24 ttgtctagcggc (SEQ ID NO: 47)gccgctagacaa (SEQ ID NO: 48) 25 tttcccttgcta (SEQ ID NO: 49)tagcaagggaaa (SEQ ID NO: 50)

Moreover, in embodiment (16), the first targeting agent and the firsttargeting agent complement can comprise a pair of oligonucleotides,wherein the pair is selected from:

Pair Sequence 1 acatcggtagtt (SEQ ID NO: 1) aactaccgatgt (SEQ ID NO: 2)3 agaagaagatcc (SEQ ID NO: 5) ggatcttcttct (SEQ ID NO: 6) 6atcattaccacc (SEQ ID NO: 11) ggtggtaatgat (SEQ ID NO: 12) 7attaacgggagc (SEQ ID NO: 13) gctcccgttaat (SEQ ID NO: 14) 8cagaggtcttaa (SEQ ID NO: 15) ttaagacctctg (SEQ ID NO: 16) 9caggtgtccatt (SEQ ID NO: 17) aatggacacctg (SEQ ID NO: 18) 11cctacgatatac (SEQ ID NO: 21) gtatatcgtagg (SEQ ID NO: 22) 12cgaatgtagagt (SEQ ID NO: 23) actctacattcg (SEQ ID NO: 24) 13cggtttgagata (SEQ ID NO: 25) tatctcaaaccg (SEQ ID NO: 26) 16gacataaagcga (SEQ ID NO: 31) tcgctttatgtc (SEQ ID NO: 32) 17gccatagtctct (SEQ ID NO: 33) agagactatggc (SEQ ID NO: 34) 18gctaattcacca (SEQ ID NO: 35) tggtgaattagc (SEQ ID NO: 36) 20gttgattctgtc (SEQ ID NO: 39) gacagaatcaac (SEQ ID NO: 40) 23ttccacttaggg (SEQ ID NO: 45) ccctaagtggaa (SEQ ID NO: 46) 25tttcccttgcta (SEQ ID NO: 49) tagcaagggaaa (SEQ ID NO: 50)

In one example of embodiment (16), the second targeting agent and thesecond targeting agent complement can include a pair ofoligonucleotides, wherein the pair is selected from:

pair # Sequence (5′-3′) 1 Acatcggtagtt (SEQ ID NO: 1)Aactaccgatgt (SEQ ID NO: 2) 2 acgtcccagttg (SEQ ID NO: 3)caactgggacgt (SEQ ID NO: 4) 3 agaagaagatcc (SEQ ID NO: 5)ggatcttcttct (SEQ ID NO: 6) 4 aggttcagtgca (SEQ ID NO: 7)tgcactgaacct (SEQ ID NO: 8) 5 atcaggatacgc (SEQ ID NO: 9)gcgtatcctgat (SEQ ID NO: 10) 6 atcattaccacc (SEQ ID NO: 11)ggtggtaatgat (SEQ ID NO: 12) 7 attaacgggagc (SEQ ID NO: 13)gctcccgttaat (SEQ ID NO: 14) 8 cagaggtcttaa (SEQ ID NO: 15)ttaagacctctg (SEQ ID NO: 16) 9 caggtgtccatt (SEQ ID NO: 17)aatggacacctg (SEQ ID NO: 18) 10 catccaatccag (SEQ ID NO: 19)ctggattggatg (SEQ ID NO: 20) 11 cctacgatatac (SEQ ID NO: 21)gtatatcgtagg (SEQ ID NO: 22) 12 cgaatgtagagt (SEQ ID NO: 23)actctacattcg (SEQ ID NO: 24) 13 cggtttgagata (SEQ ID NO: 25)tatctcaaaccg (SEQ ID NO: 26) 14 cttacaacgcca (SEQ ID NO: 27)tggcgttgtaag (SEQ ID NO: 28) 15 ctttctcggcac (SEQ ID NO: 29)gtgccgagaaag (SEQ ID NO: 30) 16 gacataaagcga (SEQ ID NO: 31)tcgctttatgtc (SEQ ID NO: 32) 17 gccatagtctct (SEQ ID NO: 33)agagactatggc (SEQ ID NO: 34) 18 gctaattcacca (SEQ ID NO: 35)tggtgaattagc (SEQ ID NO: 36) 19 ggtcgtgtttca (SEQ ID NO: 37)tgaaacacgacc (SEQ ID NO: 38) 20 gttgattctgtc (SEQ ID NO: 39)gacagaatcaac (SEQ ID NO: 40) 21 tacccggaataa (SEQ ID NO: 41)ttattccgggta (SEQ ID NO: 42) 22 tgcttgacttgg (SEQ ID NO: 43)ccaagtcaagca (SEQ ID NO: 44) 23 ttccacttaggg (SEQ ID NO: 45)ccctaagtggaa (SEQ ID NO: 46) 24 ttgtctagcggc (SEQ ID NO: 47)gccgctagacaa (SEQ ID NO: 48) 25 tttcccttgcta (SEQ ID NO: 49)tagcaagggaaa (SEQ ID NO: 50)

In a further example of embodiment (16), the second targeting agent andthe second targeting agent complement can include a pair ofoligonucleotides, wherein the pair is selected from:

Pair Sequence 1 acatcggtagtt (SEQ ID NO: 1) aactaccgatgt (SEQ ID NO: 2)3 agaagaagatcc (SEQ ID NO: 5) ggatcttcttct (SEQ ID NO: 6) 6atcattaccacc (SEQ ID NO: 11) ggtggtaatgat (SEQ ID NO: 12) 7attaacgggagc (SEQ ID NO: 13) gctcccgttaat (SEQ ID NO: 14) 8cagaggtcttaa (SEQ ID NO: 15) ttaagacctctg (SEQ ID NO: 16) 9caggtgtccatt (SEQ ID NO: 17) aatggacacctg (SEQ ID NO: 18) 11cctacgatatac (SEQ ID NO: 21) gtatatcgtagg (SEQ ID NO: 22) 12cgaatgtagagt (SEQ ID NO: 23) actctacattcg (SEQ ID NO: 24) 13cggtttgagata (SEQ ID NO: 25) tatctcaaaccg (SEQ ID NO: 26) 16gacataaagcga (SEQ ID NO: 31) tcgctttatgtc (SEQ ID NO: 32) 17gccatagtctct (SEQ ID NO: 33) agagactatggc (SEQ ID NO: 34) 18gctaattcacca (SEQ ID NO: 35) tggtgaattagc (SEQ ID NO: 36) 20gttgattctgtc (SEQ ID NO: 39) gacagaatcaac (SEQ ID NO: 40) 23ttccacttaggg (SEQ ID NO: 45) ccctaagtggaa (SEQ ID NO: 46) 25tttcccttgcta (SEQ ID NO: 49) tagcaagggaaa (SEQ ID NO: 50)

Embodiment (16) can include one or more of the following elements: thefirst targeting agent and first targeting agent complement comprise afirst oligonucleotide and a first complementary oligonucleotide,respectively, and the first oligonucleotide and the first complementaryoligonucleotide each comprise approximately 10 to 50 bases; the firsttargeting agent and first targeting agent complement comprise a firstoligonucleotide and a first complementary oligonucleotide, respectively,and the first oligonucleotide and the first complementaryoligonucleotide each comprise approximately 10 to 25 bases; the firstbinding reagent is an antibody comprising a biotin molecule and thefirst targeting agent is a first oligonucleotide comprising astreptavidin molecule and the linking complex is formed by a reactionbetween the biotin and streptavidin molecules; and/or the second bindingreagent is an antibody comprising an additional biotin molecule and thesecond targeting agent is a second oligonucleotide comprising anadditional streptavidin molecule and the linking complex is formed by areaction between the additional biotin and the additional streptavidinmolecules.

Embodiment (17): a method of conducting a binding assay for a pluralityof analytes comprising: (a) forming a first binding reagent complexcomprising a first binding reagent specific for a first analyte in theplurality of analytes and a first targeting agent, wherein the firstbinding reagent is bound to a linking agent and the first targetingagent is bound to a supplemental linking agent wherein the first bindingreagent complex is formed by a reaction between the linking agent andthe supplemental linking agent; (b) forming a second binding reagentcomplex comprising a second binding reagent specific for a secondanalyte in the plurality of analytes and a second targeting agent,wherein the second binding reagent is bound to a second linking agentand the second targeting agent is bound to a second linking agentcomplement wherein the second binding reagent complex is formed by areaction between the second linking agent and the second linking agentcomplement; (c) mixing the first and second binding reagent complexeswith two or more binding domains each linked to a first targeting agentcomplement and a second targeting agent complement, respectively, underconditions sufficient to bind the first targeting agent to the firsttargeting agent complement and the second targeting agent to the secondtargeting agent complement; (d) mixing a sample comprising the pluralityof analytes to the mixture formed in step (c); (e) adding a plurality ofadditional binding reagents to the mixture formed in step (d), whereinthe plurality of additional binding reagents includes (i) a firstdetection reagent specific for the first analyte and/or a first bindingreagent-first analyte complex; and (ii) a second detection reagentspecific for the second analyte and/or a second binding reagent-secondanalyte complex; and (f) measuring the amount of the first and secondanalytes bound to the binding domains.

Embodiment (17) can include one or more of the following features: thefirst and second binding reagents each comprise a receptor, ligand,antibody, hapten, antigen, epitope, mimitope, aptamer, or anintercalater capable of binding to the first and second analytes,respectively, e.g., the first and second binding reagents each comprisean antibody capable of binding to the first and second analyte,respectively; the first targeting agent and the first targeting agentcomplement comprise an oligonucleotide and a complementaryoligonucleotide, a receptor-ligand pair, an antigen-antibody pair, ahapten-antibody pair, an antigen-antibody pair, an epitope-antibodypair, an mimitope-antibody pair, an aptamer-target molecule pair,hybridization partners, or an intercalater-target molecule pair; thesecond targeting agent and the second targeting agent complementcomprise an oligonucleotide and a complementary oligonucleotide, areceptor-ligand pair, an antigen-antibody pair, a hapten-antibody pair,an antigen-antibody pair, an epitope-antibody pair, an mimitope-antibodypair, an aptamer-target molecule pair, hybridization partners, or anintercalater-target molecule pair, the first targeting agent and thefirst targeting agent complement comprise an oligonucleotide and acomplementary oligonucleotide; the second targeting agent and the secondtargeting agent complement comprise an oligonucleotide and acomplementary oligonucleotide; the linking complex comprises a linkingagent and a supplemental linking agent connected thereto, e.g., thelinking complex is formed by a binding interaction between (a) a thiolgroup and a maleimide or iodoacetamide; (b) an aldehyde and a hydrazide;or (c) an alkyne and an azide; the (a) the linking agent is biotin andsupplemental linking agent is streptavidin or avidin; (b) the linkingagent is streptavidin or avidin and the supplemental linking agent isbiotin; (c) the linking agent is a peptide and the supplemental linkingagent is an anti-peptide antibody; or (d) the linking agent is ananti-peptide antibody and the supplemental linking agent is a peptide.

Moreover, embodiment (17) can further include one or more of thefollowing features: each of the plurality of detection reagents comprisea detectable label, e.g., a subset of the plurality of detectionreagents comprise a detectable label; the measuring step comprisesmeasuring the presence of the detectable label in the sample via opticalabsorbance, fluorescence, phosphorescence, chemiluminescence,electrochemiluminescence, light scattering, or magnetism; the detectablelabel is an electrochemiluminescent label and the measuring stepcomprises measuring an electrochemiluminescent signal and correlatingthe signal with an amount of analyte in the sample; the two or morebinding domains are positioned on an electrode and the measuring stepfurther comprises applying a voltage waveform to the electrode togenerate electrochemiluminescence, e.g., the two or more binding domainsare located within one or more wells of a multi-well plate.

Embodiment (17) can comprise conducting a sandwich immunoassay or acompetitive immunoassay.

In embodiment (17), the first targeting agent and the first targetingagent complement comprise a pair of oligonucleotides, wherein the pairis selected from:

pair # Sequence (5′-3′) 1 Acatcggtagtt (SEQ ID NO: 1)Aactaccgatgt (SEQ ID NO: 2) 2 acgtcccagttg (SEQ ID NO: 3)caactgggacgt (SEQ ID NO: 4) 3 agaagaagatcc (SEQ ID NO: 5)ggatcttcttct (SEQ ID NO: 6) 4 aggttcagtgca (SEQ ID NO: 7)tgcactgaacct (SEQ ID NO: 8) 5 atcaggatacgc (SEQ ID NO: 9)gcgtatcctgat (SEQ ID NO: 10) 6 atcattaccacc (SEQ ID NO: 11)ggtggtaatgat (SEQ ID NO: 12) 7 attaacgggagc (SEQ ID NO: 13)gctcccgttaat (SEQ ID NO: 14) 8 cagaggtcttaa (SEQ ID NO: 15)ttaagacctctg (SEQ ID NO: 16) 9 caggtgtccatt (SEQ ID NO: 17)aatggacacctg (SEQ ID NO: 18) 10 catccaatccag (SEQ ID NO: 19)ctggattggatg (SEQ ID NO: 20) 11 cctacgatatac (SEQ ID NO: 21)gtatatcgtagg (SEQ ID NO: 22) 12 cgaatgtagagt (SEQ ID NO: 23)actctacattcg (SEQ ID NO: 24) 13 cggtttgagata (SEQ ID NO: 25)tatctcaaaccg (SEQ ID NO: 26) 14 cttacaacgcca (SEQ ID NO: 27)tggcgttgtaag (SEQ ID NO: 28) 15 ctttctcggcac (SEQ ID NO: 29)gtgccgagaaag (SEQ ID NO: 30) 16 gacataaagcga (SEQ ID NO: 31)tcgctttatgtc (SEQ ID NO: 32) 17 gccatagtctct (SEQ ID NO: 33)agagactatggc (SEQ ID NO: 34) 18 gctaattcacca (SEQ ID NO: 35)tggtgaattagc (SEQ ID NO: 36) 19 ggtcgtgtttca (SEQ ID NO: 37)tgaaacacgacc (SEQ ID NO: 38) 20 gttgattctgtc (SEQ ID NO: 39)gacagaatcaac (SEQ ID NO: 40) 21 tacccggaataa (SEQ ID NO: 41)ttattccgggta (SEQ ID NO: 42) 22 tgcttgacttgg (SEQ ID NO: 43)ccaagtcaagca (SEQ ID NO: 44) 23 ttccacttaggg (SEQ ID NO: 45)ccctaagtggaa (SEQ ID NO: 46) 24 ttgtctagcggc (SEQ ID NO: 47)gccgctagacaa (SEQ ID NO: 48) 25 tttcccttgcta (SEQ ID NO: 49)tagcaagggaaa (SEQ ID NO: 50)

In a specific example of embodiment (17), the first targeting agent andthe first targeting agent complement comprise a pair ofoligonucleotides, wherein the pair is selected from:

Pair Sequence 1 acatcggtagtt (SEQ ID NO: 1) aactaccgatgt (SEQ ID NO: 2)3 agaagaagatcc (SEQ ID NO: 5) ggatcttcttct (SEQ ID NO: 6) 6atcattaccacc (SEQ ID NO: 11) ggtggtaatgat (SEQ ID NO: 12) 7attaacgggagc (SEQ ID NO: 13) gctcccgttaat (SEQ ID NO: 14) 8cagaggtcttaa (SEQ ID NO: 15) ttaagacctctg (SEQ ID NO: 16) 9caggtgtccatt (SEQ ID NO: 17) aatggacacctg (SEQ ID NO: 18) 11cctacgatatac (SEQ ID NO: 21) gtatatcgtagg (SEQ ID NO: 22) 12cgaatgtagagt (SEQ ID NO: 23) actctacattcg (SEQ ID NO: 24) 13cggtttgagata (SEQ ID NO: 25) tatctcaaaccg (SEQ ID NO: 26) 16gacataaagcga (SEQ ID NO: 31) tcgctttatgtc (SEQ ID NO: 32) 17gccatagtctct (SEQ ID NO: 33) agagactatggc (SEQ ID NO: 34) 18gctaattcacca (SEQ ID NO: 35) tggtgaattagc (SEQ ID NO: 36) 20gttgattctgtc (SEQ ID NO: 39) gacagaatcaac (SEQ ID NO: 40) 23ttccacttaggg (SEQ ID NO: 45) ccctaagtggaa (SEQ ID NO: 46) 25tttcccttgcta (SEQ ID NO: 49) tagcaagggaaa (SEQ ID NO: 50)

In a further example of embodiment (17), the second targeting agent andthe second targeting agent complement comprise a pair ofoligonucleotides, wherein the pair is selected from:

pair # Sequence (5′-3′) 1 Acatcggtagtt (SEQ ID NO: 1)Aactaccgatgt (SEQ ID NO: 2) 2 acgtcccagttg (SEQ ID NO: 3)caactgggacgt (SEQ ID NO: 4) 3 agaagaagatcc (SEQ ID NO: 5)ggatcttcttct (SEQ ID NO: 6) 4 aggttcagtgca (SEQ ID NO: 7)tgcactgaacct (SEQ ID NO: 8) 5 atcaggatacgc (SEQ ID NO: 9)gcgtatcctgat (SEQ ID NO: 10) 6 atcattaccacc (SEQ ID NO: 11)ggtggtaatgat (SEQ ID NO: 12) 7 attaacgggagc (SEQ ID NO: 13)gctcccgttaat (SEQ ID NO: 14) 8 cagaggtcttaa (SEQ ID NO: 15)ttaagacctctg (SEQ ID NO: 16) 9 caggtgtccatt (SEQ ID NO: 17)aatggacacctg (SEQ ID NO: 18) 10 catccaatccag (SEQ ID NO: 19)ctggattggatg (SEQ ID NO: 20) 11 cctacgatatac (SEQ ID NO: 21)gtatatcgtagg (SEQ ID NO: 22) 12 cgaatgtagagt (SEQ ID NO: 23)actctacattcg (SEQ ID NO: 24) 13 cggtttgagata (SEQ ID NO: 25)tatctcaaaccg (SEQ ID NO: 26) 14 cttacaacgcca (SEQ ID NO: 27)tggcgttgtaag (SEQ ID NO: 28) 15 ctttctcggcac (SEQ ID NO: 29)gtgccgagaaag (SEQ ID NO: 30) 16 gacataaagcga (SEQ ID NO: 31)tcgctttatgtc (SEQ ID NO: 32) 17 gccatagtctct (SEQ ID NO: 33)agagactatggc (SEQ ID NO: 34) 18 gctaattcacca (SEQ ID NO: 35)tggtgaattagc (SEQ ID NO: 36) 19 ggtcgtgtttca (SEQ ID NO: 37)tgaaacacgacc (SEQ ID NO: 38) 20 gttgattctgtc (SEQ ID NO: 39)gacagaatcaac (SEQ ID NO: 40) 21 tacccggaataa (SEQ ID NO: 41)ttattccgggta (SEQ ID NO: 42) 22 tgcttgacttgg (SEQ ID NO: 43)ccaagtcaagca (SEQ ID NO: 44) 23 ttccacttaggg (SEQ ID NO: 45)ccctaagtggaa (SEQ ID NO: 46) 24 ttgtctagcggc (SEQ ID NO: 47)gccgctagacaa (SEQ ID NO: 48) 25 tttcccttgcta (SEQ ID NO: 49)tagcaagggaaa (SEQ ID NO: 50)

In yet a further example of embodiment (17), the second targeting agentand the second targeting agent complement comprise a pair ofoligonucleotides, wherein the pair is selected from:

Pair Sequence 1 acatcggtagtt (SEQ ID NO: 1) aactaccgatgt (SEQ ID NO: 2)3 agaagaagatcc (SEQ ID NO: 5) ggatcttcttct (SEQ ID NO: 6) 6atcattaccacc (SEQ ID NO: 11) ggtggtaatgat (SEQ ID NO: 12) 7attaacgggagc (SEQ ID NO: 13) gctcccgttaat (SEQ ID NO: 14) 8cagaggtcttaa (SEQ ID NO: 15) ttaagacctctg (SEQ ID NO: 16) 9caggtgtccatt (SEQ ID NO: 17) aatggacacctg (SEQ ID NO: 18) 11cctacgatatac (SEQ ID NO: 21) gtatatcgtagg (SEQ ID NO: 22) 12cgaatgtagagt (SEQ ID NO: 23) actctacattcg (SEQ ID NO: 24) 13cggtttgagata (SEQ ID NO: 25) tatctcaaaccg (SEQ ID NO: 26) 16gacataaagcga (SEQ ID NO: 31) tcgctttatgtc (SEQ ID NO: 32) 17gccatagtctct (SEQ ID NO: 33) agagactatggc (SEQ ID NO: 34) 18gctaattcacca (SEQ ID NO: 35) tggtgaattagc (SEQ ID NO: 36) 20gttgattctgtc (SEQ ID NO: 39) gacagaatcaac (SEQ ID NO: 40) 23ttccacttaggg (SEQ ID NO: 45) ccctaagtggaa (SEQ ID NO: 46) 25tttcccttgcta (SEQ ID NO: 49) tagcaagggaaa (SEQ ID NO: 50)

Embodiment (17) can include one or more of the following elements: thefirst targeting agent and the first targeting agent complement comprisea first oligonucleotide and a first complementary oligonucleotide,respectively, and the first oligonucleotide and the first complementaryoligonucleotide each comprise approximately 10 to 50 bases; the firsttargeting agent and the first targeting agent complement comprise afirst oligonucleotide and a first complementary oligonucleotide,respectively, and the first oligonucleotide and the first complementaryoligonucleotide each comprise approximately 10 to 25 bases; the firstbinding reagent is an antibody comprising a biotin molecule and thefirst targeting agent comprises a first oligonucleotide including astreptavidin molecule and the linking complex is formed by a reactionbetween the biotin and streptavidin molecules; the second bindingreagent is an antibody comprising an additional biotin molecule and thesecond targeting agent comprises a second oligonucleotide including anadditional streptavidin molecule and the linking complex is formed by areaction between the additional biotin and the additional streptavidinmolecules; the second targeting agent and the second targeting agentcomplement comprise a second oligonucleotide and a second complementaryoligonucleotide, respectively, and the second oligonucleotide and thesecond complementary oligonucleotide each comprise approximately 10 to50 bases; the second targeting agent and the second targeting agentcomplement comprise a second oligonucleotide and a second complementaryoligonucleotide, respectively, and the second oligonucleotide and thesecond complementary oligonucleotide each comprise approximately 10 to25 bases; the second binding reagent is an antibody comprising a biotinmolecule and the second targeting agent comprises a secondoligonucleotide including a streptavidin molecule and the linkingcomplex is formed by a reaction between the biotin and streptavidinmolecules; and/or the method further comprises the step of washing themixture formed in step (c) prior to mixing step (d).

Embodiment (18): a method of conducting a binding assay for a pluralityof analytes in a sample comprising: (a) forming a first binding reagentcomplex comprising a first binding reagent specific for a first analytein the plurality of analytes and a first targeting agent, wherein thefirst binding reagent is bound to a linking agent and the firsttargeting agent is bound to a supplemental linking agent wherein thefirst binding reagent complex is formed by a reaction between thelinking agent and the supplemental linking agent; (b) forming a secondbinding reagent complex comprising a second binding reagent specific fora second analyte in the plurality of analytes and a second targetingagent, wherein the second binding reagent is bound to a second linkingagent and the second targeting agent is bound to a second linking agentcomplement wherein the second binding reagent complex is formed by areaction between the second linking agent and the second linking agentcomplement; (c) mixing the first and second binding reagent complexesand the sample with two or more binding domains each linked to a firsttargeting agent complement and a second targeting agent complement,respectively, under conditions sufficient to bind the first targetingagent to the first targeting agent complement and the second targetingagent to the second targeting agent complement; (d) adding a pluralityof additional binding reagents to the mixture formed in step (c),wherein the plurality of additional binding reagents includes (i) afirst detection reagent specific for the first analyte and/or a firstbinding reagent-first analyte complex; and (ii) a second detectionreagent specific for the second analyte and/or a second bindingreagent-second analyte complex; and (e) measuring the amount of thefirst and second analytes bound to the binding domains.

In addition, embodiment (18) can include one or more of the following:the first and second binding reagents each comprise a receptor, ligand,antibody, hapten, antigen, epitope, mimitope, aptamer, or anintercalater capable of binding to the first and second analytes,respectively, first and second binding reagents each comprise anantibody capable of binding to the first and second analyte,respectively; first targeting agent and the first targeting agentcomplement comprise an oligonucleotide and a complementaryoligonucleotide, a receptor-ligand pair, an antigen-antibody pair, ahapten-antibody pair, an antigen-antibody pair, an epitope-antibodypair, an mimitope-antibody pair, an aptamer-target molecule pair,hybridization partners, or an intercalater-target molecule pair, thesecond targeting agent and the second targeting agent complementcomprise an oligonucleotide and a complementary oligonucleotide, areceptor-ligand pair, an antigen-antibody pair, a hapten-antibody pair,an antigen-antibody pair, an epitope-antibody pair, an mimitope-antibodypair, an aptamer-target molecule pair, hybridization partners, or anintercalater-target molecule pair, the first targeting agent and thefirst targeting agent complement comprise an oligonucleotide and acomplementary oligonucleotide; the second targeting agent and the secondtargeting agent complement comprise an oligonucleotide and acomplementary oligonucleotide; the linking complex comprises a linkingagent and a supplemental linking agent connected thereto, e.g., thelinking complex is formed by a binding interaction between (a) a thiolgroup and a maleimide or iodoacetamide; (b) an aldehyde and a hydrazide;or (c) an alkyne and an azide; e.g., (a) the linking agent is biotin andsupplemental linking agent is streptavidin or avidin; (b) the linkingagent is streptavidin or avidin and the supplemental linking agent isbiotin; (c) the linking agent is a peptide and the supplemental linkingagent is an anti-peptide antibody; or (d) the linking agent is ananti-peptide antibody and the supplemental linking agent is a peptide.

Still further, embodiment (18) can include one or more of the followingelements: each of the plurality of detection reagents comprise adetectable label; the plurality of detection reagents comprise adetectable label; the measuring step comprises measuring the presence ofthe detectable label in the sample via optical absorbance, fluorescence,phosphorescence, chemiluminescence, electrochemiluminescence, lightscattering, or magnetism; the detectable label is anelectrochemiluminescent label and the measuring step comprises measuringan electrochemiluminescent signal and correlating the signal with anamount of analyte in the sample; the two or more binding domains arepositioned on an electrode and the measuring step further comprisesapplying a voltage waveform to the electrode to generateelectrochemiluminescence; and/or the two or more binding domains arelocated within one or more wells of a multi-well plate.

Embodiment (18) can include conducting a sandwich immunoassay or acompetitive immunoassay.

In a specific example of embodiment (18), the first targeting agent andthe first targeting agent complement comprise a pair ofoligonucleotides, wherein the pair is selected from:

pair # Sequence (5′-3′) 1 Acatcggtagtt (SEQ ID NO: 1)Aactaccgatgt (SEQ ID NO: 2) 2 acgtcccagttg (SEQ ID NO: 3)caactgggacgt (SEQ ID NO: 4) 3 agaagaagatcc (SEQ ID NO: 5)ggatcttcttct (SEQ ID NO: 6) 4 aggttcagtgca (SEQ ID NO: 7)tgcactgaacct (SEQ ID NO: 8) 5 atcaggatacgc (SEQ ID NO: 9)gcgtatcctgat (SEQ ID NO: 10) 6 atcattaccacc (SEQ ID NO: 11)ggtggtaatgat (SEQ ID NO: 12) 7 attaacgggagc (SEQ ID NO: 13)gctcccgttaat (SEQ ID NO: 14) 8 cagaggtcttaa (SEQ ID NO: 15)ttaagacctctg (SEQ ID NO: 16) 9 caggtgtccatt (SEQ ID NO: 17)aatggacacctg (SEQ ID NO: 18) 10 catccaatccag (SEQ ID NO: 19)ctggattggatg (SEQ ID NO: 20) 11 cctacgatatac (SEQ ID NO: 21)gtatatcgtagg (SEQ ID NO: 22) 12 cgaatgtagagt (SEQ ID NO: 23)actctacattcg (SEQ ID NO: 24) 13 cggtttgagata (SEQ ID NO: 25)tatctcaaaccg (SEQ ID NO: 26) 14 cttacaacgcca (SEQ ID NO: 27)tggcgttgtaag (SEQ ID NO: 28) 15 ctttctcggcac (SEQ ID NO: 29)gtgccgagaaag (SEQ ID NO: 30) 16 gacataaagcga (SEQ ID NO: 31)tcgctttatgtc (SEQ ID NO: 32) 17 gccatagtctct (SEQ ID NO: 33)agagactatggc (SEQ ID NO: 34) 18 gctaattcacca (SEQ ID NO: 35)tggtgaattagc (SEQ ID NO: 36) 19 ggtcgtgtttca (SEQ ID NO: 37)tgaaacacgacc (SEQ ID NO: 38) 20 gttgattctgtc (SEQ ID NO: 39)gacagaatcaac (SEQ ID NO: 40) 21 tacccggaataa (SEQ ID NO: 41)ttattccgggta (SEQ ID NO: 42) 22 tgcttgacttgg (SEQ ID NO: 43)ccaagtcaagca (SEQ ID NO: 44) 23 ttccacttaggg (SEQ ID NO: 45)ccctaagtggaa (SEQ ID NO: 46) 24 ttgtctagcggc (SEQ ID NO: 47)gccgctagacaa (SEQ ID NO: 48) 25 tttcccttgcta (SEQ ID NO: 49)tagcaagggaaa (SEQ ID NO: 50)

In a another example of embodiment (18), the first targeting agent andthe first targeting agent complement comprise a pair ofoligonucleotides, wherein the pair is selected from:

Pair Sequence 1 acatcggtagtt (SEQ ID NO: 1) aactaccgatgt (SEQ ID NO: 2)3 agaagaagatcc (SEQ ID NO: 5) ggatcttcttct (SEQ ID NO: 6) 6atcattaccacc (SEQ ID NO: 11) ggtggtaatgat (SEQ ID NO: 12) 7attaacgggagc (SEQ ID NO: 13) gctcccgttaat (SEQ ID NO: 14) 8cagaggtcttaa (SEQ ID NO: 15) ttaagacctctg (SEQ ID NO: 16) 9caggtgtccatt (SEQ ID NO: 17) aatggacacctg (SEQ ID NO: 18) 11cctacgatatac (SEQ ID NO: 21) gtatatcgtagg (SEQ ID NO: 22) 12cgaatgtagagt (SEQ ID NO: 23) actctacattcg (SEQ ID NO: 24) 13cggtttgagata (SEQ ID NO: 25) tatctcaaaccg (SEQ ID NO: 26) 16gacataaagcga (SEQ ID NO: 31) tcgctttatgtc (SEQ ID NO: 32) 17gccatagtctct (SEQ ID NO: 33) agagactatggc (SEQ ID NO: 34) 18gctaattcacca (SEQ ID NO: 35) tggtgaattagc (SEQ ID NO: 36) 20gttgattctgtc (SEQ ID NO: 39) gacagaatcaac (SEQ ID NO: 40) 23ttccacttaggg (SEQ ID NO: 45) ccctaagtggaa (SEQ ID NO: 46) 25tttcccttgcta (SEQ ID NO: 49) tagcaagggaaa (SEQ ID NO: 50)

In yet another example of embodiment (18), the second targeting agentand the second targeting agent complement comprise a pair ofoligonucleotides, wherein the pair is selected from:

pair # Sequence (5′-3′) 1 Acatcggtagtt (SEQ ID NO: 1)Aactaccgatgt (SEQ ID NO: 2) 2 acgtcccagttg (SEQ ID NO: 3)caactgggacgt (SEQ ID NO: 4) 3 agaagaagatcc (SEQ ID NO: 5)ggatcttcttct (SEQ ID NO: 6) 4 aggttcagtgca (SEQ ID NO: 7)tgcactgaacct (SEQ ID NO: 8) 5 atcaggatacgc (SEQ ID NO: 9)gcgtatcctgat (SEQ ID NO: 10) 6 atcattaccacc (SEQ ID NO: 11)ggtggtaatgat (SEQ ID NO: 12) 7 attaacgggagc (SEQ ID NO: 13)gctcccgttaat (SEQ ID NO: 14) 8 cagaggtcttaa (SEQ ID NO: 15)ttaagacctctg (SEQ ID NO: 16) 9 caggtgtccatt (SEQ ID NO: 17)aatggacacctg (SEQ ID NO: 18) 10 catccaatccag (SEQ ID NO: 19)ctggattggatg (SEQ ID NO: 20) 11 cctacgatatac (SEQ ID NO: 21)gtatatcgtagg (SEQ ID NO: 22) 12 cgaatgtagagt (SEQ ID NO: 23)actctacattcg (SEQ ID NO: 24) 13 cggtttgagata (SEQ ID NO: 25)tatctcaaaccg (SEQ ID NO: 26) 14 cttacaacgcca (SEQ ID NO: 27)tggcgttgtaag (SEQ ID NO: 28) 15 ctttctcggcac (SEQ ID NO: 29)gtgccgagaaag (SEQ ID NO: 30) 16 gacataaagcga (SEQ ID NO: 31)tcgctttatgtc (SEQ ID NO: 32) 17 gccatagtctct (SEQ ID NO: 33)agagactatggc (SEQ ID NO: 34) 18 gctaattcacca (SEQ ID NO: 35)tggtgaattagc (SEQ ID NO: 36) 19 ggtcgtgtttca (SEQ ID NO: 37)tgaaacacgacc (SEQ ID NO: 38) 20 gttgattctgtc (SEQ ID NO: 39)gacagaatcaac (SEQ ID NO: 40) 21 tacccggaataa (SEQ ID NO: 41)ttattccgggta (SEQ ID NO: 42) 22 tgcttgacttgg (SEQ ID NO: 43)ccaagtcaagca (SEQ ID NO: 44) 23 ttccacttaggg (SEQ ID NO: 45)ccctaagtggaa (SEQ ID NO: 46) 24 ttgtctagcggc (SEQ ID NO: 47)gccgctagacaa (SEQ ID NO: 48) 25 tttcccttgcta (SEQ ID NO: 49)tagcaagggaaa (SEQ ID NO: 50)

In a further example of embodiment (18), the second targeting agent andthe second targeting agent complement comprise a pair ofoligonucleotides, wherein the pair is selected from:

Pair Sequence 1 acatcggtagtt (SEQ ID NO: 1) aactaccgatgt (SEQ ID NO: 2)3 agaagaagatcc (SEQ ID NO: 5) ggatcttcttct (SEQ ID NO: 6) 6atcattaccacc (SEQ ID NO: 11) ggtggtaatgat (SEQ ID NO: 12) 7attaacgggagc (SEQ ID NO: 13) gctcccgttaat (SEQ ID NO: 14) 8cagaggtcttaa (SEQ ID NO: 15) ttaagacctctg (SEQ ID NO: 16) 9caggtgtccatt (SEQ ID NO: 17) aatggacacctg (SEQ ID NO: 18) 11cctacgatatac (SEQ ID NO: 21) gtatatcgtagg (SEQ ID NO: 22) 12cgaatgtagagt (SEQ ID NO: 23) actctacattcg (SEQ ID NO: 24) 13cggtttgagata (SEQ ID NO: 25) tatctcaaaccg (SEQ ID NO: 26) 16gacataaagcga (SEQ ID NO: 31) tcgctttatgtc (SEQ ID NO: 32) 17gccatagtctct (SEQ ID NO: 33) agagactatggc (SEQ ID NO: 34) 18gctaattcacca (SEQ ID NO: 35) tggtgaattagc (SEQ ID NO: 36) 20gttgattctgtc (SEQ ID NO: 39) gacagaatcaac (SEQ ID NO: 40) 23ttccacttaggg (SEQ ID NO: 45) ccctaagtggaa (SEQ ID NO: 46) 25tttcccttgcta (SEQ ID NO: 49) tagcaagggaaa (SEQ ID NO: 50)

Embodiment (18) can include one or more of the following features: thefirst targeting agent and the first targeting agent complement comprisea first oligonucleotide and a first complementary oligonucleotide,respectively, and the first oligonucleotide and the first complementaryoligonucleotide each comprise approximately 10 to 50 bases; the firsttargeting agent and the first targeting agent complement comprise afirst oligonucleotide and a first complementary oligonucleotide,respectively, and the first oligonucleotide and the first complementaryoligonucleotide each comprise approximately 10 to 25 bases; the firstbinding reagent is an antibody comprising a biotin molecule and thefirst targeting agent comprises a first oligonucleotide including astreptavidin molecule and the linking complex is formed by a reactionbetween the biotin and streptavidin molecules; the second bindingreagent is an antibody comprising an additional biotin molecule and thesecond targeting agent comprises a second oligonucleotide including anadditional streptavidin molecule and the linking complex is formed by areaction between the additional biotin and the additional streptavidinmolecules; the second targeting agent and the second targeting agentcomplement comprise a second oligonucleotide and a second complementaryoligonucleotide, respectively, and the second oligonucleotide and thesecond complementary oligonucleotide each comprise approximately 10 to50 bases; the second targeting agent and the second targeting agentcomplement comprise a second oligonucleotide and a second complementaryoligonucleotide, respectively, and the second oligonucleotide and thesecond complementary oligonucleotide each comprise approximately 10 to25 bases; the second binding reagent is an antibody comprising a biotinmolecule and the second targeting agent comprises a secondoligonucleotide including a streptavidin molecule and the linkingcomplex is formed by a reaction between the biotin and streptavidinmolecules; and/or the method further comprises the step of washing themixture formed in step (c) prior to mixing step (d).

Embodiment (19): a kit comprising: (a) a multi-well plate comprising aplurality of discrete binding domains each comprising a first and secondoligonucleotide, respectively; (b) in a separate vial, container, orcompartment, a set of targeting reagents comprising a firstoligonucleotide complement bound to a linking agent and a secondoligonucleotide complement bound to a second linking agent, wherein (i)the first oligonucleotide and the first oligonucleotide complementcomprise a first pair of oligonucleotides, and (ii) the secondoligonucleotide and the second oligonucleotide complement comprise asecond pair of oligonucleotides, wherein the first and second pair ofoligonucleotides, respectively, are selected from:

pair # Sequence (5′-3′) 1 Acatcggtagtt (SEQ ID NO: 1)Aactaccgatgt (SEQ ID NO: 2) 2 acgtcccagttg (SEQ ID NO: 3)caactgggacgt (SEQ ID NO: 4) 3 agaagaagatcc (SEQ ID NO: 5)ggatcttcttct (SEQ ID NO: 6) 4 aggttcagtgca (SEQ ID NO: 7)tgcactgaacct (SEQ ID NO: 8) 5 atcaggatacgc (SEQ ID NO: 9)gcgtatcctgat (SEQ ID NO: 10) 6 atcattaccacc (SEQ ID NO: 11)ggtggtaatgat (SEQ ID NO: 12) 7 attaacgggagc (SEQ ID NO: 13)gctcccgttaat (SEQ ID NO: 14) 8 cagaggtcttaa (SEQ ID NO: 15)ttaagacctctg (SEQ ID NO: 16) 9 caggtgtccatt (SEQ ID NO: 17)aatggacacctg (SEQ ID NO: 18) 10 catccaatccag (SEQ ID NO: 19)ctggattggatg (SEQ ID NO: 20) 11 cctacgatatac (SEQ ID NO: 21)gtatatcgtagg (SEQ ID NO: 22) 12 cgaatgtagagt (SEQ ID NO: 23)actctacattcg (SEQ ID NO: 24) 13 cggtttgagata (SEQ ID NO: 25)tatctcaaaccg (SEQ ID NO: 26) 14 cttacaacgcca (SEQ ID NO: 27)tggcgttgtaag (SEQ ID NO: 28) 15 ctttctcggcac (SEQ ID NO: 29)gtgccgagaaag (SEQ ID NO: 30) 16 gacataaagcga (SEQ ID NO: 31)tcgctttatgtc (SEQ ID NO: 32) 17 gccatagtctct (SEQ ID NO: 33)agagactatggc (SEQ ID NO: 34) 18 gctaattcacca (SEQ ID NO: 35)tggtgaattagc (SEQ ID NO: 36) 19 ggtcgtgtttca (SEQ ID NO: 37)tgaaacacgacc (SEQ ID NO: 38) 20 gttgattctgtc (SEQ ID NO: 39)gacagaatcaac (SEQ ID NO: 40) 21 tacccggaataa (SEQ ID NO: 41)ttattccgggta (SEQ ID NO: 42) 22 tgcttgacttgg (SEQ ID NO: 43)ccaagtcaagca (SEQ ID NO: 44) 23 ttccacttaggg (SEQ ID NO: 45)ccctaagtggaa (SEQ ID NO: 46) 24 ttgtctagcggc (SEQ ID NO: 47)gccgctagacaa (SEQ ID NO: 48) 25 tttcccttgcta (SEQ ID NO: 49)tagcaagggaaa (SEQ ID NO: 50)

Embodiment (20): a kit comprising: (a) a multi-well plate comprising aplurality of discrete binding domains each comprising a first and secondoligonucleotide, respectively; (b) in a separate vial, container, orcompartment, a set of targeting reagents comprising a firstoligonucleotide complement bound to a linking agent and a secondoligonucleotide complement bound to a second linking agent, wherein (i)the first oligonucleotide and the first oligonucleotide complementcomprise a first pair of oligonucleotides, and (ii) the secondoligonucleotide and the second oligonucleotide complement comprise asecond pair of oligonucleotides, wherein the first and second pair ofoligonucleotides, respectively, are selected from:

Pair Sequence 1 acatcggtagtt (SEQ ID NO: 1) aactaccgatgt (SEQ ID NO: 2)3 agaagaagatcc (SEQ ID NO: 5) ggatcttcttct (SEQ ID NO: 6) 6atcattaccacc (SEQ ID NO: 11) ggtggtaatgat (SEQ ID NO: 12) 7attaacgggagc (SEQ ID NO: 13) gctcccgttaat (SEQ ID NO: 14) 8cagaggtcttaa (SEQ ID NO: 15) ttaagacctctg (SEQ ID NO: 16) 9caggtgtccatt (SEQ ID NO: 17) aatggacacctg (SEQ ID NO: 18) 11cctacgatatac (SEQ ID NO: 21) gtatatcgtagg (SEQ ID NO: 22) 12cgaatgtagagt (SEQ ID NO: 23) actctacattcg (SEQ ID NO: 24) 13cggtttgagata (SEQ ID NO: 25) tatctcaaaccg (SEQ ID NO: 26) 16gacataaagcga (SEQ ID NO: 31) tcgctttatgtc (SEQ ID NO: 32) 17gccatagtctct (SEQ ID NO: 33) agagactatggc (SEQ ID NO: 34) 18gctaattcacca (SEQ ID NO: 35) tggtgaattagc (SEQ ID NO: 36) 20gttgattctgtc (SEQ ID NO: 39) gacagaatcaac (SEQ ID NO: 40) 23ttccacttaggg (SEQ ID NO: 45) ccctaagtggaa (SEQ ID NO: 46) 25tttcccttgcta (SEQ ID NO: 49) tagcaagggaaa (SEQ ID NO: 50)

Embodiments (19) and (20) can further include (b) one or more additionalcontainers, vessels or compartments comprising: (i) a first bindingreagent comprising a linking agent, wherein the first binding reagent isspecific for a first analyte in the sample, (ii) a first targeting agentcomplement comprising a supplemental linking agent, provided in aseparate container, vessel or compartment or as a component of

(b)(i), (iii) a second binding reagent comprising a second linkingagent, wherein the second binding reagent is specific for a secondanalyte in the sample, and (iv) a second targeting agent complementcomprising a second linking agent complement, provided in a separatecontainer, vessel or compartment or as a component of (b)(iii).

Embodiment (21): a kit for measuring a plurality of different analytesin a sample, the kit comprising: (a) a container, vessel or compartmentcomprising on a solid support a first targeting agent immobilized to afirst region of the solid support and a second targeting agentimmobilized to a second region of the solid support; and (b) one or moreadditional containers, vessels or compartments comprising: (i) a firstbinding reagent comprising a linking agent, wherein the first bindingreagent is specific for a first analyte in the sample, (ii) a firsttargeting agent complement comprising a supplemental linking agent,provided in a separate container, vessel or compartment or as acomponent of (b)(i), (iii) a second binding reagent comprising a secondlinking agent, wherein the second binding reagent is specific for asecond analyte in the sample, and (iv) a second targeting agentcomplement comprising a second linking agent complement, provided in aseparate container, vessel or compartment or as a component of (b)(iii).

Embodiment (22): a kit for measuring a plurality of different analytesin a sample, the kit comprising: (a) a container, vessel or compartmentcomprising on a solid support a first targeting agent immobilized to afirst region of the solid support and a second targeting agentimmobilized to a second region of the solid support; and (b) four ormore additional containers, vessels or compartments comprising: (i) afirst container comprising a first binding reagent comprising a firstlinking agent, wherein the first binding reagent is specific for a firstanalyte in the sample, (ii) a second container comprising a firsttargeting agent complement comprising a supplemental linking agent,provided in a separate container, vessel or compartment, (iii) a thirdcontainer comprising a second binding reagent comprising a secondlinking agent, wherein the second binding reagent is specific for asecond analyte in the sample, and (iv) a fourth container comprising asecond targeting agent complement comprising a second linking agentcomplement, provided in a separate container, vessel or compartment.

Embodiment (23): a kit for measuring a plurality of different analytesin a sample, the kit comprising: (a) a container, vessel or compartmentcomprising on a solid support a first targeting agent immobilized to afirst region of the solid support and a second targeting agentimmobilized to a second region of the solid support; and (b) two or moreadditional containers, vessels or compartments comprising: (i) a firstcontainer comprising a first binding reagent comprising a first linkingagent, wherein the first binding reagent is specific for a first analytein the sample, and a first targeting agent complement comprising a firstlinking agent complement, and (ii) a second container comprising asecond binding reagent comprising a second linking agent, wherein thesecond binding reagent is specific for a second analyte in the sample,and a second targeting agent complement comprising a second linkingagent complement.

Embodiment (24): a kit comprising (a) a multi-well plate comprising aplurality of discrete binding domains each comprising a first and secondoligonucleotide, respectively, each of the first and secondoligonucleotides are selected from:

Sequence (5′-3′) Acatcggtagtt (SEQ ID NO: 1) Aactaccgatgt (SEQ ID NO: 2)acgtcccagttg (SEQ ID NO: 3) caactgggacgt (SEQ ID NO: 4)agaagaagatcc (SEQ ID NO: 5) ggatcttcttct (SEQ ID NO: 6)aggttcagtgca (SEQ ID NO: 7) tgcactgaacct (SEQ ID NO: 8)atcaggatacgc (SEQ ID NO: 9) gcgtatcctgat (SEQ ID NO: 10)atcattaccacc (SEQ ID NO: 11) ggtggtaatgat (SEQ ID NO: 12)attaacgggagc (SEQ ID NO: 13) gctcccgttaat (SEQ ID NO: 14)cagaggtcttaa (SEQ ID NO: 15) ttaagacctctg (SEQ ID NO: 16)caggtgtccatt (SEQ ID NO: 17) aatggacacctg (SEQ ID NO: 18)catccaatccag (SEQ ID NO: 19) ctggattggatg (SEQ ID NO: 20)cctacgatatac (SEQ ID NO: 21) gtatatcgtagg (SEQ ID NO: 22)cgaatgtagagt (SEQ ID NO: 23) actctacattcg (SEQ ID NO: 24)cggtttgagata (SEQ ID NO: 25) tatctcaaaccg (SEQ ID NO: 26)cttacaacgcca (SEQ ID NO: 27) tggcgttgtaag (SEQ ID NO: 28)ctttctcggcac (SEQ ID NO: 29) gtgccgagaaag (SEQ ID NO: 30)gacataaagcga (SEQ ID NO: 31) tcgctttatgtc (SEQ ID NO: 32)gccatagtctct (SEQ ID NO: 33) agagactatggc (SEQ ID NO: 34)gctaattcacca (SEQ ID NO: 35) tggtgaattagc (SEQ ID NO: 36)ggtcgtgtttca (SEQ ID NO: 37) tgaaacacgacc (SEQ ID NO: 38)gttgattctgtc (SEQ ID NO: 39) gacagaatcaac (SEQ ID NO: 40)tacccggaataa (SEQ ID NO: 41) ttattccgggta (SEQ ID NO: 42)tgcttgacttgg (SEQ ID NO: 43) ccaagtcaagca (SEQ ID NO: 44)ttccacttaggg (SEQ ID NO: 45) ccctaagtggaa (SEQ ID NO: 46)ttgtctagcggc (SEQ ID NO: 47) gccgctagacaa (SEQ ID NO: 48)tttcccttgcta (SEQ ID NO: 49) tagcaagggaaa (SEQ ID NO: 50)

The kit of embodiment (24) can include one or more of the followingsequences:

Sequence acatcggtagtt (SEQ ID NO: 1) aactaccgatgt (SEQ ID NO: 2)agaagaagatcc (SEQ ID NO: 5) ggatcttcttct (SEQ ID NO: 6)atcattaccacc (SEQ ID NO: 11) ggtggtaatgat (SEQ ID NO: 12)attaacgggagc (SEQ ID NO: 13) gctcccgttaat (SEQ ID NO: 14)cagaggtcttaa (SEQ ID NO: 15) ttaagacctctg (SEQ ID NO: 16)caggtgtccatt (SEQ ID NO: 17) aatggacacctg (SEQ ID NO: 18)cctacgatatac (SEQ ID NO: 21) gtatatcgtagg (SEQ ID NO: 22)cgaatgtagagt (SEQ ID NO: 23) actctacattcg (SEQ ID NO: 24)cggtttgagata (SEQ ID NO: 25) tatctcaaaccg (SEQ ID NO: 26)gacataaagcga (SEQ ID NO: 31) tcgctttatgtc (SEQ ID NO: 32)gccatagtctct (SEQ ID NO: 33) agagactatggc (SEQ ID NO: 34)gctaattcacca (SEQ ID NO: 35) tggtgaattagc (SEQ ID NO: 36)gttgattctgtc (SEQ ID NO: 39) gacagaatcaac (SEQ ID NO: 40)ttccacttaggg (SEQ ID NO: 45) ccctaagtggaa (SEQ ID NO: 46)tttcccttgcta (SEQ ID NO: 49) tagcaagggaaa (SEQ ID NO: 50)

Embodiment (24) can further include instructions for use of themulti-well plate in a method of conducting a binding assay for aplurality of analytes, the method comprising the steps of: (a) forming afirst binding reagent complex comprising a first binding reagentspecific for a first analyte in the plurality of analytes and the firstoligonucleotide, wherein the first binding reagent is bound to a linkingagent and the first oligonucleotide is bound to a supplemental linkingagent wherein the first binding reagent complex is formed by a reactionbetween the linking agent and the supplemental linking agent; (b)forming a second binding reagent complex comprising a second bindingreagent specific for a second analyte in the plurality of analytes andthe second oligonucleotide, wherein the second binding reagent is boundto a second linking agent and the second oligonucleotide is bound to asecond linking agent complement wherein the second binding reagentcomplex is formed by a reaction between the second linking agent and thesecond linking agent complement; (c) mixing the first and second bindingreagent complexes with the two or more binding domains each linked to afirst oligonucleotide complement and a second oligonucleotidecomplement, respectively, under conditions sufficient to bind the firstoligonucleotide to the first oligonucleotide complement and the secondoligonucleotide to the second oligonucleotide complement; (d) mixing asample comprising the plurality of analytes to the mixture formed instep (c); (e) adding a plurality of additional binding reagents to themixture formed in step (d), wherein the plurality of additional bindingreagents includes (i) a first detection reagent specific for the firstanalyte and/or a first binding reagent-first analyte complex; and (ii) asecond detection reagent specific for the second analyte and/or a secondbinding reagent-second analyte complex; and (f) measuring the amount ofthe first and second analytes bound to the binding domains.

In addition, embodiment (24) can include one or more of the followingelements: the first and second binding reagents each comprise areceptor, ligand, antibody, hapten, antigen, epitope, mimitope, aptamer,or an intercalater capable of binding to the first and second analytes,respectively, e.g., the first and second binding reagents each comprisean antibody capable of binding to the first and second analyte,respectively; the first targeting agent and the first targeting agentcomplement comprise an oligonucleotide and a complementaryoligonucleotide, a receptor-ligand pair, an antigen-antibody pair, ahapten-antibody pair, an antigen-antibody pair, an epitope-antibodypair, an mimitope-antibody pair, an aptamer-target molecule pair,hybridization partners, or an intercalater-target molecule pair, thesecond targeting agent and the second targeting agent complementcomprise an oligonucleotide and a complementary oligonucleotide, areceptor-ligand pair, an antigen-antibody pair, a hapten-antibody pair,an antigen-antibody pair, an epitope-antibody pair, an mimitope-antibodypair, an aptamer-target molecule pair, hybridization partners, or anintercalater-target molecule pair, the first targeting agent and thefirst targeting agent complement comprise an oligonucleotide and acomplementary oligonucleotide; the second targeting agent and the secondtargeting agent complement comprise an oligonucleotide and acomplementary oligonucleotide; the linking complex comprises a linkingagent and a supplemental linking agent connected thereto, e.g., thelinking complex is formed by a binding interaction between (a) a thiolgroup and a maleimide or iodoacetamide; (b) an aldehyde and a hydrazide;or (c) an alkyne and an azide; or the (a) the linking agent is biotinand supplemental linking agent is streptavidin or avidin; (b) thelinking agent is streptavidin or avidin and the supplemental linkingagent is biotin; (c) the linking agent is a peptide and the supplementallinking agent is an anti-peptide antibody; or (d) the linking agent isan anti-peptide antibody and the supplemental linking agent is apeptide.

In addition, embodiment (24) can further comprise, in a separate vial,container, or compartment, a plurality of detection reagents. Forexample, the plurality of detection reagents can comprise a detectablelabel, e.g., a subset of the plurality of detection reagents comprise adetectable label. In one example, the detectable label is anelectrochemiluminescent label. The two or more binding domains can bepositioned on an electrode. Optionally, the two or more binding domainsare located within one or more wells of a multi-well plate.

In a specific example of embodiment (24), the first targeting agent andthe first targeting agent complement comprise a pair ofoligonucleotides, wherein the pair is selected from:

Pair Sequence 1 acatcggtagtt (SEQ ID NO: 1) aactaccgatgt (SEQ ID NO: 2)3 agaagaagatcc (SEQ ID NO: 5) ggatcttcttct (SEQ ID NO: 6) 6atcattaccacc (SEQ ID NO: 11) ggtggtaatgat (SEQ ID NO: 12) 7attaacgggagc (SEQ ID NO: 13) gctcccgttaat (SEQ ID NO: 14) 8cagaggtcttaa (SEQ ID NO: 15) ttaagacctctg (SEQ ID NO: 16) 9caggtgtccatt (SEQ ID NO: 17) aatggacacctg (SEQ ID NO: 18) 11cctacgatatac (SEQ ID NO: 21) gtatatcgtagg (SEQ ID NO: 22) 12cgaatgtagagt (SEQ ID NO: 23) actctacattcg (SEQ ID NO: 24) 13cggtttgagata (SEQ ID NO: 25) tatctcaaaccg (SEQ ID NO: 26) 16gacataaagcga (SEQ ID NO: 31) tcgctttatgtc (SEQ ID NO: 32) 17gccatagtctct (SEQ ID NO: 33) agagactatggc (SEQ ID NO: 34) 18gctaattcacca (SEQ ID NO: 35) tggtgaattagc (SEQ ID NO: 36) 20gttgattctgtc (SEQ ID NO: 39) gacagaatcaac (SEQ ID NO: 40) 23ttccacttaggg (SEQ ID NO: 45) ccctaagtggaa (SEQ ID NO: 46) 25tttcccttgcta (SEQ ID NO: 49) tagcaagggaaa (SEQ ID NO: 50)

In embodiment (24), the second targeting agent and the second targetingagent complement comprise a pair of oligonucleotides, wherein the pairis selected from:

pair # Sequence (5′-3′) 1 Acatcggtagtt (SEQ ID NO: 1)Aactaccgatgt (SEQ ID NO: 2) 2 acgtcccagttg (SEQ ID NO: 3)caactgggacgt (SEQ ID NO: 4) 3 agaagaagatcc (SEQ ID NO: 5)ggatcttcttct (SEQ ID NO: 6) 4 aggttcagtgca (SEQ ID NO: 7)tgcactgaacct (SEQ ID NO: 8) 5 atcaggatacgc (SEQ ID NO: 9)gcgtatcctgat (SEQ ID NO: 10) 6 atcattaccacc (SEQ ID NO: 11)ggtggtaatgat (SEQ ID NO: 12) 7 attaacgggagc (SEQ ID NO: 13)gctcccgttaat (SEQ ID NO: 14) 8 cagaggtcttaa (SEQ ID NO: 15)ttaagacctctg (SEQ ID NO: 16) 9 caggtgtccatt (SEQ ID NO: 17)aatggacacctg (SEQ ID NO: 18) 10 catccaatccag (SEQ ID NO: 19)ctggattggatg (SEQ ID NO: 20) 11 cctacgatatac (SEQ ID NO: 21)gtatatcgtagg (SEQ ID NO: 22) 12 cgaatgtagagt (SEQ ID NO: 23)actctacattcg (SEQ ID NO: 24) 13 cggtttgagata (SEQ ID NO: 25)tatctcaaaccg (SEQ ID NO: 26) 14 cttacaacgcca (SEQ ID NO: 27)tggcgttgtaag (SEQ ID NO: 28) 15 ctttctcggcac (SEQ ID NO: 29)gtgccgagaaag (SEQ ID NO: 30) 16 gacataaagcga (SEQ ID NO: 31)tcgctttatgtc (SEQ ID NO: 32) 17 gccatagtctct (SEQ ID NO: 33)agagactatggc (SEQ ID NO: 34) 18 gctaattcacca (SEQ ID NO: 35)tggtgaattagc (SEQ ID NO: 36) 19 ggtcgtgtttca (SEQ ID NO: 37)tgaaacacgacc (SEQ ID NO: 38) 20 gttgattctgtc (SEQ ID NO: 39)gacagaatcaac (SEQ ID NO: 40) 21 tacccggaataa (SEQ ID NO: 41)ttattccgggta (SEQ ID NO: 42) 22 tgcttgacttgg (SEQ ID NO: 43)ccaagtcaagca (SEQ ID NO: 44) 23 ttccacttaggg (SEQ ID NO: 45)ccctaagtggaa (SEQ ID NO: 46) 24 ttgtctagcggc (SEQ ID NO: 47)gccgctagacaa (SEQ ID NO: 48) 25 tttcccttgcta (SEQ ID NO: 49)tagcaagggaaa (SEQ ID NO: 50)

In another specific example of embodiment (24), the first targetingagent and the first targeting agent complement comprise a pair ofoligonucleotides, wherein the pair is selected from:

Pair Sequence 1 acatcggtagtt (SEQ ID NO: 1) aactaccgatgt (SEQ ID NO: 2)3 agaagaagatcc (SEQ ID NO: 5) ggatcttcttct (SEQ ID NO: 6) 6atcattaccacc (SEQ ID NO: 11) ggtggtaatgat (SEQ ID NO: 12) 7attaacgggagc (SEQ ID NO: 13) gctcccgttaat (SEQ ID NO: 14) 8cagaggtcttaa (SEQ ID NO: 15) ttaagacctctg (SEQ ID NO: 16) 9caggtgtccatt (SEQ ID NO: 17) aatggacacctg (SEQ ID NO: 18) 11cctacgatatac (SEQ ID NO: 21) gtatatcgtagg (SEQ ID NO: 22) 12cgaatgtagagt (SEQ ID NO: 23) actctacattcg (SEQ ID NO: 24) 13cggtttgagata (SEQ ID NO: 25) tatctcaaaccg (SEQ ID NO: 26) 16gacataaagcga (SEQ ID NO: 31) tcgctttatgtc (SEQ ID NO: 32) 17gccatagtctct (SEQ ID NO: 33) agagactatggc (SEQ ID NO: 34) 18gctaattcacca (SEQ ID NO: 35) tggtgaattagc (SEQ ID NO: 36) 20gttgattctgtc (SEQ ID NO: 39) gacagaatcaac (SEQ ID NO: 40) 23ttccacttaggg (SEQ ID NO: 45) ccctaagtggaa (SEQ ID NO: 46) 25tttcccttgcta (SEQ ID NO: 49) tagcaagggaaa (SEQ ID NO: 50)

Embodiment (24) can also include one or more of the following: the firsttargeting agent and the first targeting agent complement comprise afirst oligonucleotide and a first complementary oligonucleotide,respectively, and the first oligonucleotide and the first complementaryoligonucleotide each comprise approximately 10 to 50 bases; the firsttargeting agent and the first targeting agent complement comprise afirst oligonucleotide and a first complementary oligonucleotide,respectively, and the first oligonucleotide and the first complementaryoligonucleotide each comprise approximately 10 to 25 bases; the firstbinding reagent is an antibody comprising a biotin molecule and thefirst targeting agent comprises a first oligonucleotide including astreptavidin molecule and the linking complex is formed by a reactionbetween the biotin and streptavidin molecules; the second bindingreagent is an antibody comprising an additional biotin molecule and thesecond targeting agent comprises a second oligonucleotide including anadditional streptavidin molecule and the linking complex is formed by areaction between the additional biotin and the additional streptavidinmolecules; the second targeting agent and the second targeting agentcomplement comprise a second oligonucleotide and a second complementaryoligonucleotide, respectively, and the second oligonucleotide and thesecond complementary oligonucleotide each comprise approximately 10 to50 bases; the second targeting agent and the second targeting agentcomplement comprise a second oligonucleotide and a second complementaryoligonucleotide, respectively, and the second oligonucleotide and thesecond complementary oligonucleotide each comprise approximately 10 to25 bases; and/or the second binding reagent is an antibody comprising abiotin molecule and the second targeting agent comprises a secondoligonucleotide including a streptavidin molecule and the linkingcomplex is formed by a reaction between the biotin and streptavidinmolecules.

In any one or more of the preceding embodiments, the amount of the firstbinding reagent on the second binding domain can be <1% the amount ofthe first binding reagent on the first binding domain.

Embodiment (25): a kit comprising: a multi-well plate having one or morecopies of an oligonucleotide array within at least one well(s) of theplate, the array is positioned on a plurality of binding domains,wherein at least 4 of the binding domains have immobilized thereon adifferent oligonucleotide sequence selected from:

(SEQ ID NO: 1) Acatcggtagtt (SEQ ID NO: 2) Aactaccgatgt (SEQ ID NO: 3)acgtcccagttg (SEQ ID NO: 4) caactgggacgt (SEQ ID NO: 5) agaagaagatcc(SEQ ID NO: 6) ggatcttcttct (SEQ ID NO: 7) aggttcagtgca (SEQ ID NO: 8)tgcactgaacct (SEQ ID NO: 9) atcaggatacgc (SEQ ID NO: 10) gcgtatcctgat(SEQ ID NO: 11) atcattaccacc (SEQ ID NO: 12) ggtggtaatgat(SEQ ID NO: 13) attaacgggagc (SEQ ID NO: 14) gctcccgttaat(SEQ ID NO: 15) cagaggtcttaa (SEQ ID NO: 16) ttaagacctctg(SEQ ID NO: 17) caggtgtccatt (SEQ ID NO: 18) aatggacacctg(SEQ ID NO: 19) catccaatccag (SEQ ID NO: 20) ctggattggatg(SEQ ID NO: 21) cctacgatatac (SEQ ID NO: 22) gtatatcgtagg(SEQ ID NO: 23) cgaatgtagagt (SEQ ID NO: 24) actctacattcg(SEQ ID NO: 25) cggtttgagata (SEQ ID NO: 26) tatctcaaaccg(SEQ ID NO: 27) cttacaacgcca (SEQ ID NO: 28) tggcgttgtaag(SEQ ID NO: 29) ctttctcggcac (SEQ ID NO: 30) gtgccgagaaag(SEQ ID NO: 31) gacataaagcga (SEQ ID NO: 32) tcgctttatgtc(SEQ ID NO: 33) gccatagtctct (SEQ ID NO: 34) agagactatggc(SEQ ID NO: 35) gctaattcacca (SEQ ID NO: 36) tggtgaattagc(SEQ ID NO: 37) ggtcgtgtttca (SEQ ID NO: 38) tgaaacacgacc(SEQ ID NO: 39) gttgattctgtc (SEQ ID NO: 40) gacagaatcaac(SEQ ID NO: 41) tacccggaataa (SEQ ID NO: 42) ttattccgggta(SEQ ID NO: 43) tgcttgacttgg (SEQ ID NO: 44) ccaagtcaagca(SEQ ID NO: 45) ttccacttaggg (SEQ ID NO: 46) ccctaagtggaa(SEQ ID NO: 47) ttgtctagcggc (SEQ ID NO: 48) gccgctagacaa(SEQ ID NO: 49) tttcccttgcta (SEQ ID NO: 50) tagcaagggaaa

wherein each of the different oligonucleotide sequences is modified witha linking agent.

Embodiment (26): a kit comprising: a multi-well plate having one or morecopies of an oligonucleotide array within at least one well(s) of theplate, the array is positioned on a plurality of binding domains,wherein at least 4 of the binding domains have immobilized thereon adifferent oligonucleotide sequence selected from:

Pair Sequence 1 acatcggtagtt (SEQ ID NO: 1) aactaccgatgt (SEQ ID NO: 2)3 agaagaagatcc (SEQ ID NO: 5) ggatcttcttct (SEQ ID NO: 6) 6atcattaccacc (SEQ ID NO: 11) ggtggtaatgat (SEQ ID NO: 12) 7attaacgggagc (SEQ ID NO: 13) gctcccgttaat (SEQ ID NO: 14) 8cagaggtcttaa (SEQ ID NO: 15) ttaagacctctg (SEQ ID NO: 16) 9caggtgtccatt (SEQ ID NO: 17) aatggacacctg (SEQ ID NO: 18) 11cctacgatatac (SEQ ID NO: 21) gtatatcgtagg (SEQ ID NO: 22) 12cgaatgtagagt (SEQ ID NO: 23) actctacattcg (SEQ ID NO: 24) 13cggtttgagata (SEQ ID NO: 25) tatctcaaaccg (SEQ ID NO: 26) 16gacataaagcga (SEQ ID NO: 31) tcgctttatgtc (SEQ ID NO: 32) 17gccatagtctct (SEQ ID NO: 33) agagactatggc (SEQ ID NO: 34) 18gctaattcacca (SEQ ID NO: 35) tggtgaattagc (SEQ ID NO: 36) 20gttgattctgtc (SEQ ID NO: 39) gacagaatcaac (SEQ ID NO: 40) 23ttccacttaggg (SEQ ID NO: 45) ccctaagtggaa (SEQ ID NO: 46) 25tttcccttgcta (SEQ ID NO: 49) tagcaagggaaa (SEQ ID NO: 50)

Embodiments (25) and (26) can include one or more of the followingelements: the linking agent comprises a biotin, streptavidin, avidin,amino group, thiol group, aldehyde group, hydrazide group, azide group,alkyne group, maleimide group or iodoacetamide group; the linking agentis streptavidin; the kit further includes, in one or more separatevials, containers, or compartments, (a) a set of binding reagent pairs,wherein each binding reagent of the set is specific for an analyte in asample comprising a plurality of analytes, wherein optionally, a bindingreagent pair within the set comprises a first binding reagent comprisinga supplemental linking agent, and still further optionally the kitfurther includes, in one or more separate vials, containers, orcompartments, a supplemental linking agent. In one example, the bindingreagent is an antibody.

The kits of the preceding embodiments, e.g., embodiments (25) and (26),can further include, in one or more separate vials, containers, orcompartments, a labeling kit, as well as one or more additional reagentscomprising: an assay buffer, diluent, read buffer, or combinationsthereof. The labeling kit can include, in one or more separate vials,containers, or compartments, SULFO-TAG™ NHS ester, LC-biotin NHS ester,a spin column, a labeling buffer solution, ECL read buffer, assay andantibody buffers, assay and antibody diluents, or combinations thereof.

In embodiments (25) and (26), the array comprises at least 7 bindingdomains, or at least 10 binding domains, or at least 16 binding domains,or at least 25 binding domains. Moreover, the plate can include at least24 wells, or at least 96 wells, or at least 384 wells. The array canalso include at least 10 oligonucleotides, or at least 16oligonucleotides, or at least 25 oligonucleotides.

In a specific example of any one of the preceding embodiments, thenumber of binding reagent pairs in the set is equivalent to the numberof binding domains in the array, or the number of binding reagent pairsin the set is less than the number of binding domains in the array.

BRIEF DESCRIPTION OF DRAWINGS

The accompanying drawings are provided to illustrate rather than limitthe scope of the invention.

FIGS. 1(a)-(d) illustrate an assay format comprising the directconjugation of binding reagents, A′, B′, and C′, to a plurality ofbinding domains, X, Y, and Z, respectively, via reactions betweentargeting agents, A″, B″, and C″, and targeting agent complements, A′″,B′″, and C′″, respectively. The targeting agents are attached to bindingreagents, A′, B′, and C′, on a series of binding domains, X, Y, and Z,to form binding reagent complexes, A_(RC), B_(RC), and C_(RC),respectively (panel (b)), which react with analytes A, B, and C,respectively. The presence of analytes A, B, and C on the solid supportis detected by the addition of labeled detection reagents, A*, B*, andC*, which react with analytes, A, B, and C, respectively (panel (c)).FIG. 1(d) is an enlarged view of the binding reagent complexes formed onbinding domains X, Y, and Z, binding reagent complexes A_(RC), B_(RC),and C_(RC), respectively.

FIGS. 2(a)-(d) illustrates an assay format comprising the directconjugation of binding reagents, e.g., antibodies, A′, B′, and C′, to aplurality of binding domains, X, Y, and Z, respectively, via reactionsbetween oligonucleotide targeting agents, A″, B″, and C″, andoligonucleotide targeting agent complements, A′″, B′″, and C′″,respectively. The oligonucleotide targeting agents are attached tobinding reagents, A′, B′, and C′, on a series of binding domains, X, Y,and Z, to form binding reagent complexes, A_(RC), B_(RC), and C_(RC),respectively (panel (b)), which react with analytes A, B, and C,respectively. The presence of analytes A, B, and C on the solid supportis detected by the addition of labeled detection reagents, A*, B*, andC*, which react with analytes, A, B, and C, respectively (panel (c)).FIG. 2(d) is an enlarged view of the binding reagent complexes formed onbinding domains X, Y, and Z, binding reagent complexes A_(RC), B_(RC),and C_(RC), respectively.

FIGS. 3(a)-(e) illustrates an assay format involving the conjugation ofa plurality of binding reagents, A′, B′, and C′, to a plurality ofbinding domains, X, Y, and Z, respectively, via a series of linkingcomplexes. As shown in panel (a), binding reagents A′, B′, and C′ areattached to linking agents, L_(A), L_(B), and L_(C), while targetingagents A″, B″, and C′ are attached to supplemental linking agents,L_(A)′, L_(B)′, and L_(C)′, respectively. Binding reagents A′, B′, andC′ are mixed with targeting agents to form binding reagent complexes,A_(RC), B_(RC), and C_(RC), respectively. The binding reagent complexesformed in panel (a) are mixed with a plurality of binding domains, X, Y,and Z, to which targeting agent complements, A′″, B′″, and C′″ are bound(panel (b)) to adhere the binding reagent complexes to the bindingdomains (see panel (c)). A sample comprising analytes A, B, and C, isadded to the mixture, and simultaneously or sequentially, a set ofdetection reagents, A*, B*, and C* are also added to detect analytesbound to the binding domains. FIG. 3(e) is an enlarged view of thebinding reagent complexes formed on binding domains X, Y, and Z, bindingreagent complexes A_(RC), B_(RC), and C_(RC), respectively.

FIGS. 4(a)-(e) illustrates an assay format involving the conjugation ofa plurality of binding reagents, A′, B′, and C′, to a plurality ofbinding domains, X, Y, and Z, respectively, via a series of linkingcomplexes. As shown in panel (a), binding reagents A′, B′, and C′ areattached to linking agents, L_(A), L_(B), and L_(C), whileoligonucleotide targeting agents A″, B″, and C′ are attached tosupplemental linking agents, L_(A)′, La′, and L_(C)′, respectively.Binding reagents A′, B′, and C′ are mixed with oligonucleotide targetingagents to form binding reagent complexes, A_(RC), B_(RC), and C_(RC),respectively. The binding reagent complexes formed in panel (a) aremixed with a plurality of binding domains, X, Y, and Z, to whicholigonucleotide targeting agent complements, A′″, B′″, and C′″ are bound(panel (b)) to adhere the binding reagent complexes to the bindingdomains (see panel (c)). A sample comprising analytes A, B, and C, isadded to the mixture, and simultaneously or sequentially, a set ofdetection reagents, A*, B*, and C* are also added to detect analytesbound to the binding domains. FIG. 4(e) is an enlarged view of thebinding reagent complexes formed on binding domains X, Y, and Z, bindingreagent complexes A_(RC), B_(RC), and C_(RC), respectively.

FIGS. 5(a)-(d) illustrate various combinations of reagents that can beused in the assay format of the invention. In FIG. 5(a), all reagentsare mixed with the sample in a single step, whereas in FIG. 5(b), allreagents are mixed and then added to the surface, and in 5(c), modifiedbinding reagents and modified targeting agent complements are mixed,added to a surface bearing a plurality of binding domains each includinga targeting agent, and the surface-mixture is mixed with the sample anddetection reagents in one step or two (one step addition of sample anddetection reagents is shown in FIG. 5(c) and the sequential addition ofsample and detection reagents is shown in FIG. 5(d).

FIGS. 6(a)-(f) show various modified surfaces, e.g., particles andsubstrates bearing a plurality of binding domains that can be used inthe assay format of the invention.

FIG. 7 shows a procedure to chemically modify an antibody withsulfo-SMCC to yield a maleimide-activated antibody which can beconjugated to an oligonucleotide via a thiol group on theoligonucleotide.

FIGS. 8(a)-(g) show the results of a direct assay format on a 7-plexchemokine panel and a 10-plex TH1/TH2 panel.

FIGS. 9(a)-(c) show a procedure for the production and use of amulti-well assay plate in an indirect assay format using biotinylatedcapture antibodies with oligonucleotide modified with streptavidinmolecules.

FIGS. 10(a)-(c) show a procedure for the production and use of amulti-well assay plate in an indirect assay format using biotinylatedcapture antibodies, neat streptavidin, and biotinylatedoligonucleotides.

FIGS. 11(a)-(g) show a comparison of LOD values for three differentoligonucleotide-mediated assay formats for a cytokine B panel.

DETAILED DESCRIPTION OF THE INVENTION

Unless otherwise defined herein, scientific and technical terms used inconnection with the present invention shall have the meanings that arecommonly understood by those of ordinary skill in the art. Further,unless otherwise required by context, singular terms shall includepluralities and plural terms shall include the singular. The articles“a” and “an” are used herein to refer to one or to more than one (i.e.,to at least one) of the grammatical object of the article. By way ofexample, “an element” means one element or more than one element.

The present invention provides flexible solid phase binding assayformats that allow a user or manufacturer to configure an assay based onspecific user requirements. The methods and kits described hereinprovide a flexible platform for creating a multiplexed binding assay fora plurality of target analytes. With a support including a plurality ofbinding domains bearing a series of generic targeting agent complements,it is possible to configure a multiplexed assay for any set of analytes.One only needs to select which analytes will be evaluated in whichbinding domain and pair the appropriate binding reagents and targetingagents with each selected binding domain. Using this platform, a usercan build a personalized assay panel.

Such flexible multiplexed assay formats can be achieved using themethods and products disclosed herein. For example, a method ofconducting a multiplexed binding assay for a plurality of analytes ofinterest can be implemented using the following steps:

(a) combining, in one or more steps, the following components:

-   -   (i) a sample comprising a first analyte of interest and a second        analyte of interest,    -   (ii) a first targeting agent immobilized on a first binding        domain,    -   (iii) a first targeting agent complement connected to a linking        agent, wherein the first targeting agent complement is a binding        partner of the first targeting agent,    -   (iv) a first binding reagent connected to a supplemental linking        agent, wherein the first binding reagent is a binding partner of        the first analyte,    -   (v) a second targeting agent immobilized on a second binding        domain,    -   (vi) a second targeting agent complement connected to a linking        agent, wherein the second targeting agent complement is a        binding partner of the second targeting agent,    -   (vii) a second binding reagent connected to a supplemental        linking agent, wherein the second binding reagent is a binding        partner of the second analyte, and    -   (viii) optionally, at least two copies of a bridging agent,    -   wherein, if the bridging agent is omitted, each linking agent is        a binding partner of the supplemental linking agent, or if the        bridging agent is included, the bridging agent has a first        binding site for one of the linking agents and an additional        binding site for one of the supplemental linking agents;

(b) forming

-   -   (i) a first binding complex on the first binding domain        comprising the first targeting agent, the first targeting agent        complement, the first binding reagent and the first analyte, and    -   (ii) a second binding complex on the second binding domain        comprising the second targeting agent, the second targeting        agent complement, the second binding reagent and the second        analyte, and

(c) measuring the amount of the first and second analytes on the firstand second binding domains, respectively.

In one embodiment, if a bridging agent is not used, the method includes(a) combining components (i)-(vii) in one or more steps, (b) forming thefirst and second binding complexes on the first and second bindingdomains, respectively, and (c) measuring the amount of the first andsecond analytes on the first and second binding domains, respectively.For example, the first targeting agent complement and the first bindingreagent can be provided as a pre-bound first targeting complex includingthe first targeting agent complement and the first binding reagentlinked through a binding interaction between the linking agent andsupplemental linking agent; and likewise, the second targeting agentcomplement and the second binding reagent can be provided as a pre-boundsecond targeting complex comprising the second targeting agentcomplement and the second binding reagent linked through a bindinginteraction between the linking agent and supplemental linking agent. Inthis embodiment, the first targeting complex can be provided pre-boundto the first targeting agent immobilized on the first binding domain;and likewise, the second targeting complex can be provided pre-bound tothe second targeting agent immobilized on the second binding domain.When the first and second binding reagents are provided in pre-boundtargeting complexes, the combining step may further includes combiningthe first and second targeting complexes with the sample to form amixture thereof, binding the first analyte to the first binding reagentin the first targeting complex and binding the second analyte to thesecond binding reagent in the second targeting complex, contacting amixture of the first and second targeting complexes bound to first andsecond analytes, respectively, with the first and second bindingdomains. The binding complexes on the first and second domains arethereby formed by binding the first targeting complex to the firsttargeting agent on the first binding domain and binding the secondtargeting complex to the second targeting agent on the second bindingdomain. Moreover, the combining step can further include combining thefirst and second targeting complexes with the sample; and binding thefirst analyte to the first binding reagent in the first targetingcomplex and binding the second analyte to the second binding reagent inthe second targeting complex.

In another alternative embodiment, the combining step (a) includes thesteps of combining, in a first volume of liquid, said first targetingagent complement, said first binding reagent and, if used, said bridgingreagent and linking said first targeting agent complement and said firstbinding reagent through their attached linking agents to form a firsttargeting complex; and combining, in a second volume of liquid, saidsecond targeting agent complement, said second binding reagent and, ifused, said bridging reagent and linking said second targeting agentcomplex complement and said second binding reagent through theirattached linking agents to form a second targeting complex. In thisembodiment, the combining step (a) can also include the steps ofcombining said first and second targeting complexes, contacting thecombination of said first and second targeting complexes with said firstand second binding domains, and binding said first targeting complex tosaid first targeting agent on said first binding domain and binding saidsecond targeting complex to said second targeting agent on said secondbinding domain. In this embodiment, the combining step further includescombining the combination of the first and second targeting complexeswith the sample and binding the first analyte to the first bindingreagent in the first targeting complex and binding the second analyte tothe second binding reagent in the second targeting complex. The firstand second targeting complexes can be combined with the sample prior tocontacting the first and second targeting complexes with the first andsecond binding domains; the first and second targeting complexes can becombined with the sample after contacting the first and second targetingcomplexes with the first and second binding domains; or the first andsecond targeting complexes can be combined with the sample and contactedwith the first and second binding domains at the same time.

If a bridging agent is included in the method, then the linking agentand supplemental linking agents each bind to the bridging agent, and thecombining step therefore brings those elements attached to the linkingagents and supplemental linking agents together. For example, thecombining step (a) includes combining, in a first volume of liquid, (xi)the first targeting agent complement, the first binding reagent and thebridging reagent and further includes forming the first targetingcomplex by linking the first targeting agent complement and the firstbinding reagent through a bridging complex including the linking agentbound to the bridging agent to which the supplemental linking agent isbound. Combining step (a) also includes combining, in a second volume ofliquid, (xii) the second targeting agent complement, the second bindingreagent and the bridging reagent and further includes forming the secondtargeting complex by linking the second targeting agent complement andthe second binding reagent through a bridging complex including thelinking agent bound to the bridging agent to which the supplementallinking agent is bound. In this embodiment, the combining step (a) canalso include combining (xiii) the first and second targeting complexes,and combining the combination of the first and second targetingcomplexes with the first and second binding domains, and binding thefirst targeting complex on the first binding domain and binding thesecond targeting complex on the second binding domain. In thisembodiment, the combining step further includes combining (xiv) thefirst and second targeting complexes with the sample and binding thefirst analyte to the first binding reagent in the first targetingcomplex and binding the second analyte to the second binding reagent inthe second targeting complex The first and second targeting complexescan be combined with the sample prior to contacting the first and secondtargeting complexes with the first and second binding domains; the firstand second targeting complexes can be combined with the sample aftercontacting the first and second targeting complexes with the first andsecond binding domains; or the first and second targeting complexes canbe combined with the sample and contacted with the first and secondbinding domains at the same time.

The methods described herein can be used to multiplex a plurality ofanalytes of interest in a sample. In this regard, the sample containsone or more additional analytes of interest and for each additionalanalyte of interest, the combining step (a) further comprises combining,in one or more steps, (ix) an additional targeting agent immobilized onan additional binding domain, an additional targeting agent complementconnected to a linking agent, and an additional binding reagentconnected to a supplemental linking agent, and (x) an additional bindingcomplex on the additional binding domain comprising the additionaltargeting agent, the additional targeting agent complement, theadditional binding reagent and the additional analyte; the forming step(b) further comprises forming (iii) an additional binding complex on theadditional binding domain comprising the additional targeting agent, theadditional targeting agent complement, the additional binding reagentand the additional analyte; and the measurement in step (c) furthercomprises measuring the amount of the additional analyte on theadditional binding domain.

In a specific embodiment, the invention includes a method of conductinga binding assay for a plurality of analytes comprising (a) contacting asample with two or more binding domains linked to at least a first andsecond binding reagent that each bind a first and second analyte,respectively, of the plurality of analytes to form complexes comprisingthe first analyte bound to the first binding reagent and the secondanalyte bound to the second binding reagent, wherein (x) the firstbinding domain comprises a first binding reagent complex comprising (i)a first targeting agent bound to the first binding domain and to a firsttargeting agent complement; and (ii) the first binding reagent bound tothe first targeting agent complement via a linking complex; and (y) thesecond binding domain comprises a second binding reagent complexcomprising (i) a second targeting agent bound to the second bindingdomain and to a second targeting agent complement; and (ii) the secondbinding reagent bound to the second targeting agent complement via alinking complex; (b) contacting the first and second binding reagentcomplexes with a plurality of detection reagents comprising a firstdetection reagent that binds the first analyte or a complex comprisingthe first analyte, and a second detection reagent that binds the secondanalyte or a complex comprising the second analyte; and (c) measuringthe amount of the first and second analytes bound to the two or morebinding domains.

In another specific embodiment, the method includes: (a) forming a firstbinding reagent complex comprising a first binding reagent specific fora first analyte in the plurality of analytes and a first targetingagent, wherein the first binding reagent is bound to a linking agent andthe first targeting agent is bound to a supplemental linking agentwherein the first binding reagent complex is formed by a reactionbetween the linking agent and the supplemental linking agent; (b)forming a second binding reagent complex comprising a second bindingreagent specific for a second analyte in the plurality of analytes and asecond targeting agent, wherein the second binding reagent is bound to asecond linking agent and the second targeting agent is bound to a secondlinking agent complement wherein the second binding reagent complex isformed by a reaction between the second linking agent and the secondlinking agent complement; (c) mixing the first and second bindingreagent complexes with two or more binding domains each linked to afirst targeting agent complement and a second targeting agentcomplement, respectively, under conditions sufficient to bind the firsttargeting agent to the first targeting agent complement and the secondtargeting agent to the second targeting agent complement; (d) mixing asample comprising the plurality of analytes to the mixture formed instep (c); (e) adding a plurality of additional binding reagents to themixture formed in step (d), wherein the plurality of additional bindingreagents includes (i) a first detection reagent specific for the firstanalyte and/or a first binding reagent-first analyte complex; and (ii) asecond detection reagent specific for the second analyte and/or a secondbinding reagent-second analyte complex; and (f) measuring the amount ofthe first and second analytes bound to the binding domains.

A further specific embodiment includes (a) forming a first bindingreagent complex comprising a first binding reagent specific for a firstanalyte in the plurality of analytes and a first targeting agent,wherein the first binding reagent is bound to a linking agent and thefirst targeting agent is bound to a supplemental linking agent whereinthe first binding reagent complex is formed by a reaction between thelinking agent and the supplemental linking agent; (b) forming a secondbinding reagent complex comprising a second binding reagent specific fora second analyte in the plurality of analytes and a second targetingagent, wherein the second binding reagent is bound to a second linkingagent and the second targeting agent is bound to a second linking agentcomplement wherein the second binding reagent complex is formed by areaction between the second linking agent and the second linking agentcomplement; (c) mixing the first and second binding reagent complexesand the sample with two or more binding domains each linked to a firsttargeting agent complement and a second targeting agent complement,respectively, under conditions sufficient to bind the first targetingagent to the first targeting agent complement and the second targetingagent to the second targeting agent complement; (d) adding a pluralityof additional binding reagents to the mixture formed in step (c),wherein the plurality of additional binding reagents includes (i) afirst detection reagent specific for the first analyte and/or a firstbinding reagent-first analyte complex; and (ii) a second detectionreagent specific for the second analyte and/or a second bindingreagent-second analyte complex; and (e) measuring the amount of thefirst and second analytes bound to the binding domains.

Specific embodiments of the method of the present invention areillustrated in FIGS. 3-6. FIGS. 1 and 2 illustrate a direct assay methodthat does not involve a linking complex. These figures are provided forcomparative purposes. FIG. 1 illustrates a direct multiplexed assay foranalytes A, B, and C. Binding reagents specific for these analytes, A′,B′, and C′, respectively, are attached to targeting agents, A″, B″, andC″. A solution including these binding reagents attached to theircorresponding targeting agents is mixed with a solid phase to whichtargeting agent complements A′″, B′″, and C′″, respectively are bound toa series of discrete binding domains. The binding reagents are adsorbedto the surface to form binding reagent complexes, A_(RC), B_(RC), andC_(RC), each binding reagent affixed via the targeting agent complementsto a discrete binding domain on the surface. The surface is contactedwith a sample comprising analytes A, B, and C, as well as detectionbinding reagents, A*, B*, and C*, which are capable of binding toanalytes A, B, and C, respectively, and/or a complex comprising thoseanalytes. The detection binding reagents include a detectable label.Alternatively, the surface is contacted with a sample comprising theplurality of analytes and subsequently contacted with a mixture ofdetection binding reagents. Once the detection binding reagents arebound to the surface, and optionally, the surface is washed to removeunbound reagents, the presence of each analyte is detected via thedetection reagents bound to each discrete binding domain. FIG. 2illustrates a specific embodiment of FIG. 1 involving the use ofantibodies as binding reagents and oligonucleotide-complementaryoligonucleotide pairs as targeting agent/targeting agent complementpairs. It will be evident to the skilled artisan that the direct methodsillustrated in FIGS. 1 and 2 are not configurable by the user. Eachindividual binding domain includes a predetermined targeting agentcomplement, such that only a single binding reagent-targeting agent canbind to a single binding domain in the array.

FIGS. 3-4 illustrate particular embodiments of the instant inventionthat offer the user optimal flexibility in a user-defined assayconfiguration. FIG. 3 illustrates an indirect binding format foranalytes A, B, and C, incorporating a series of linking complexes thatallow the user to tailor the assay for his/her needs. FIG. 3(a)-(b)illustrates a general approach for making the targeting complexes of theinvention: a series of solutions are formed that include one of thebinding reagents (A′, B′, and C′) bound to a linking agents (L_(A),L_(B), and L_(C), respectively). The solutions also include thecorresponding targeting agents, (A″ for A′, B″ for B′, and C″ for C′),bound to a supplemental linking agent (L_(A)′, L_(B)′, and L_(C)′,respectively). The solutions are mixed to form the mixture of bindingreagent-linking complex-targeting agent complexes shown in panel (b). Anadvantage of this approach is that it does not require the linkingreagents for each targeting complex to be non-cross reactive and, infact, allows the linking agents to be used in each targeting complex(i.e., L_(A)=L_(B)=L_(C) and L_(A)′=L_(B)′=L_(C)′). In one embodimentillustrated in FIG. 3(c)-(e) the mixture of binding reagent-linkingcomplex-targeting agent complexes are mixed with a surface comprising aplurality of discrete binding domains to which targeting agentcomplements, A′″, B′″, and C′″ are bound. The binding reagent-linkingcomplex-targeting agent complexes are adsorbed to form binding reagentcomplexes, A_(RC), B_(RC), and C_(RC) as shown in panel (c). An expandedview of the binding reagent complexes is shown in FIG. 3(e). The surfaceis contacted with a sample comprising analytes A, B, and C, as well asdetection binding reagents, A*, B*, and C*, which are capable of bindingto analytes A, B, and C, respectively, and/or a complex comprising thoseanalytes. The detection binding reagents include a detectable label.Alternatively, the surface is contacted with a sample comprising theplurality of analytes and subsequently contacted with a mixture ofdetection binding reagents. Once the detection binding reagents arebound to the surface, and optionally, the surface is washed to removeunbound reagents, the presence of each analyte is detected via thedetection reagents bound to each discrete binding domain (panel 3(d)).FIG. 4 illustrates a specific embodiment of FIG. 3 involving the use ofantibodies as binding reagents and oligonucleotide-complementaryoligonucleotide pairs as targeting agent/targeting agent complementpairs. As noted for FIG. 3, the linking agents for each binding reagentmay be the same and the linking agent complements for each targetingagent may be the same.

The skilled artisan will readily appreciate that various permutations ofthe assay format depicted in FIGS. 3-4 are possible. Certain preferredembodiments are depicted in FIG. 5(a)-(c). For example, all of thereagents, i.e., binding reagents modified by supplemental linkingagents, targeting agent complements modified by linking agents,detection reagents and sample, can be mixed together with the surfacebearing targeting agent-modified binding domains in a single step toform the complexes shown in FIGS. 3(d) and 4(d), optionally washed, andanalyzed for the presence of analytes A, B, and C, bound to the surface(FIG. 5(a)). Alternatively, binding reagents modified by supplementallinking agents, and targeting agents modified by linking agents can bemixed in a single step, added to the surface having targetingagent-modified binding domains in a subsequent step, sample anddetection reagents are added, and analyzed in a final step (FIG. 5(b)).In yet another embodiment, binding reagents modified by supplementallinking agents, and targeting agent complements modified by linkingagents can be mixed, added to the surface bearing targeting agents indiscrete binding domains, mixed with sample, and then detection reagentsare added (FIG. 5(c)). Individual analyte solutions can be added to eachbinding domain sequentially or simultaneously in a single mixture, andlikewise, individual detection reagents can be added to each bindingdomain sequentially or simultaneously in a single mixture. Any surfacebinding step can optionally be followed by a washing step to remove anyunbound components of the assay before proceeding to the next step.

The invention also provides kits, components, and consumables that canbe used to practice the methods described herein. The followingmaterials/methods are used in the instant invention.

(i) Samples/Analytes

Examples of samples that may be analyzed by the methods of the presentinvention include, but are not limited to food samples (including foodextracts, food homogenates, beverages, etc.), environmental samples(e.g., soil samples, environmental sludges, collected environmentalaerosols, environmental wipes, water filtrates, etc.), industrialsamples (e.g., starting materials, products or intermediates from anindustrial production process), human clinical samples, veterinarysamples and other samples of biological origin. Biological samples thatmay be analyzed include, but are not limited to, feces, mucosal swabs,physiological fluids and/or samples containing suspensions of cells.Specific examples of biological samples include blood, serum, plasma,feces, mucosal swabs, tissue aspirates, tissue homogenates, cellcultures and cell culture supernatants (including cultures of eukaryoticand prokaryotic cells), urine, saliva, sputum, and cerebrospinal fluid.

Analytes that may be measured using the methods of the inventioninclude, but are not limited to proteins, toxins, nucleic acids,microorganisms, viruses, cells, fungi, spores, carbohydrates, lipids,glycoproteins, lipoproteins, polysaccharides, drugs, hormones, steroids,nutrients, metabolites and any modified derivative of the abovemolecules, or any complex comprising one or more of the above moleculesor combinations thereof. The level of an analyte of interest in a samplemay be indicative of a disease or disease condition or it may simplyindicate whether the patient was exposed to that analyte.

The assays of the present invention may be used to determine theconcentration of one or more, e.g., two or more analytes in a sample.Thus, two or more analytes may be measured in the same sample. Panels ofanalytes that can be measured in the same sample include, for example,panels of assays for analytes or activities associated with a diseasestate or physiological conditions. Certain such panels include panels ofcytokines and/or their receptors (e.g., one or more of TNF-alpha,TNF-beta, IL1-alpha, IL1-beta, IL2, IL4A, IL6, IL-10, IL-12, IFN-y,etc.), growth factors and/or their receptors (e.g., one or more of EGF,VGF, TGF, VEGF, etc.), drugs of abuse, therapeutic drugs, vitamins,pathogen specific antibodies, auto-antibodies (e.g., one or moreantibodies directed against the Sm, RNP, SS-A, SS-alpha, J0-1, andScl-70 antigens), allergen-specific antibodies, tumor markers (e.g., oneor more of CEA, PSA, CA-125 II, CA 15-3, CA 19-9, CA 72-4, CYFRA 21-1,NSE, AFP, etc.), markers of cardiac disease including congestive heartdisease and/or acute myocardial infarction (e.g., one or more ofTroponin T, Troponin I, myoglobin, CKMB, myeloperoxidase, glutathioneperoxidase, β-natriuretic protein (BNP), alpha-natriuretic protein(ANP), endothelin, aldosterone, C-reactive protein (CRP), etc.), markersassociated with hemostasis (e.g., one or more of Fibrin monomer,D-dimer, thrombin-antithrombin complex, prothrombin fragments 1 & 2,anti-Factor Xa, etc.), markers of acute viral hepatitis infection (e.g.,one or more of IgM antibody to hepatitis A virus, IgM antibody tohepatitis B core antigen, hepatitis B surface antigen, antibody tohepatitis C virus, etc.), markers of Alzheimers Disease (alpha-amyloid,beta-amyloid, Aβ42, Aβ 40, Aβ 38, Aβ39, Aβ37, Aβ 34, tau-protein, etc.),markers of osteoporosis (e.g., one or more of cross-linked NorC-telopeptides, total deoxypyridinoline, free deoxypyridinoline,osteocalcin, alkaline phosphatase, C-terminal propeptide of type Icollagen, bone-specific alkaline phosphatase, etc.), markers offertility state or fertility associated disorders (e.g., one or more ofEstradiol, progesterone, follicle stimulating hormone (FSH), lutenizinghormone (LH), prolactin, hCG, testosterone, etc.), markers of thyroiddisorders (e.g., one or more of thyroid stimulating hormone (TSH), TotalT3, Free T3, Total T4, Free T4, and reverse T3), and markers ofprostrate cancer (e.g., one or more of total PSA, free PSA, complexedPSA, prostatic acid phosphatase, creatine kinase, etc.). Certainembodiments of invention include measuring, e.g., one or more, two ormore, four or more or 10 or more analytes associated with a specificdisease state or physiological condition (e.g., analytes groupedtogether in a panel, such as those listed above; e.g., a panel usefulfor the diagnosis of thyroid disorders may include e.g., one or more ofthyroid stimulating hormone (TSH), Total T3, Free T3, Total T4, Free T4,and reverse T3).

The methods of the present invention are designed to allow detection ofa wide variety of biological and biochemical agents, as described above.In one embodiment, the methods may be used to detect pathogenic and/orpotentially pathogenic virus, bacteria and toxins including biologicalwarfare agents (“BWAs”) in a variety of relevant clinical andenvironmental matrices, including and without limitation, blood, sputum,stool, filters, swabs, etc. A non-limiting list of pathogens and toxinsthat may be analyzed (alone or in combination) using the methods of thepresent invention is Bacillus anthracis (anthrax), Yersinia pestis(plague), Vibrio cholerae (cholera), Francisella tularensis (tularemia),Brucella spp. (Brucellosis), Coxiella burnetii (Q fever), listeria,salmonella, shigella, V. cholera, Chlamydia trachomatis, Burkholderiapseudomallei, orthopox viruses including variola virus (smallpox), viralencephalitis, Venezuelan equine encephalitis virus (VEE), western equineencephalitis virus (WEE), eastern equine encephalitis virus (EEE),Alphavirus, viral hemorrhagic fevers, Arenaviridae, Bunyaviridae,Filoviridae, Flaviviridae, Ebola virus, staphylococcal enterotoxins,ricin, botulinum toxins (A, B, E), Clostridium botulinum, mycotoxin,Fusarium, Myrotecium, Cephalosporium, Trichoderma, Verticimonosporium,Stachybotrys, glanders, wheat fungus, Bacillus globigii, Serratiamarcescens, yellow rain, trichothecene mycotoxins, Salmonellatyphimurium, aflatoxin, Xenopsylla cheopis, Diamanus montanus, alastrim,monkeypox, Arenavirus, Hantavirus, Lassa fever, Argentine hemorrhagicfevers, Bolivian hemorrhagic fevers, Rift Valley fever virus,Crimean-Congo virus, Hanta virus, Marburg hemorrhagic fevers, yellowfever virus, dengue fever viruses, influenza (including human and animalstrains including H5N1 avian influenza, influenza A, influenza A, H1specific, influenza A, H3 specific, influenza A, H5 specific, influenzaA, 2009-H1N1 specific, influenza B), RSV, human immunodeficiency virusesI and II (HIV I and II), hepatitis A, hepatitis B, hepatitis C,hepatitis (non-A, B or C), Enterovirus, Epstein-Barr virus,Cytomegalovirus, herpes simplex viruses, Chlamydia trachomatis,Neisseria gonorrheae, Trichomonas vaginalis, human papilloma virus,Treponema pallidum, Streptococcus pneumonia, Borellia burgdorferi,Haemophilus influenzae, Mycoplasma pneumoniae, Chlamydophila pneumoniae,Legionella pneumophila, Staphylococcus aureus, StaphylococcusEnterotoxin B (SEB), Abrin, Shiga Toxin 1, Shiga Toxin 2, Moraxellacatarrhalis, Streptococcus pyogenes, Clostridium difficile, Neisseriameningitidis, Klebsiella pneumoniae, Mycobacterium tuberculosis, Group Astreptococcus, E. Coli O157, coronavirus, Coxsackie A virus, rhinovirus,parainfluenza virus, respiratory syncytial virus (RSV), metapneumovirus,vaccinia, and adenovirus.

(ii) Binding Reagents

The skilled artisan in the field of binding assays will readilyappreciate the scope of binding agents and companion binding partnersthat may be used in the present methods. A non-limiting list of suchpairs include (in either order) oligonucleotides and complements,receptor/ligand pairs, antibodies/antigens, natural or syntheticreceptor/ligand pairs, amines and carbonyl compounds (i.e., bindingthrough the formation of a Schiff's base), hapten/antibody pairs,antigen/antibody pairs, epitope/antibody pairs, mimitope/antibody pairs,aptamer/target molecule pairs, hybridization partners, andintercalater/target molecule pairs.

In a preferred embodiment, the binding assays of the methods of thepresent invention employ antibodies or other receptor proteins asbinding reagents. The term “antibody” includes intact antibody molecules(including hybrid antibodies assembled by in vitro re-association ofantibody subunits), antibody fragments and recombinant proteinconstructs comprising an antigen binding domain of an antibody (asdescribed, e.g., in Porter, R. R. and Weir, R. C. J. Cell Physiol., 67(Suppl); 51-64 (1966) and Hochman, 1. Inbar, D. and Givol, D.Biochemistry 12: 1130 (1973)), as well as antibody constructs that havebeen chemically modified, e.g., by the introduction of a detectablelabel.

iii) Targeting Agents, Linking Agents & Bridging Agents

Binding reagents are linked to components that enable their attachmentto each other and/or to solid phases, directly or indirectly. Thesecomponents are referred to herein as targeting agents and linkingagents. As used herein, targeting agents and their complements are usedto adhere a binding reagent to a surface or support, whereas linkingagents and supplemental linking agents are used to attach a bindingreagent to a targeting agent, directly or indirectly through a bridgingagent, if one is used.

In one embodiment, a targeting agent and its complement comprise a firstoligonucleotide and a complementary oligonucleotide, a receptor-ligandpair, an antigen-antibody pair, a hapten-antibody pair, anepitope-antibody pair, a mimetope-antibody pair, an aptamer-targetmolecule pair, hybridization partners, or an intercalator-targetmolecule pair. The targeting agents and complements used in an assay areselected such that the targeting agents and complements associated witha binding reagent for an analyte (for example, a first analyte) measuredby the assay are substantially non-cross-reactive with the targetingagents and complements associated with the binding reagents for theother analytes measured by the assay (for example, a second analyte).Accordingly, in an assay of the invention, the binding of a bindingreagent to its associated binding domain (through its associatedtargeting agent and targeting agent complement) should be substantiallygreater than its binding to binding domains associated with otheranalytes (and presenting different targeting agent complements).Preferably the cross-reactivity for the binding of binding reagents foran analyte to binding domains associated with other analytes relative tothe binding to the correct binding domain is <1%, more preferably <0.1%and more preferably <0.01%. In a preferred embodiment, the targetingagent/targeting agent complement comprise a pair of oligonucleotidesincluding complementary sequences and the targeting agent and itscomplement are contacted under conditions sufficient to hybridize thetargeting agent to its complement.

The preferred length is approximately 5 to 100 bases, preferably,approximately, 10 to 50 bases, and more preferably approximately 10 to25 bases. In addition, the targeting oligonucleotides sequences need notbe identical in length and in certain embodiments it may be beneficialto provide one targeting oligonucleotide sequence that is longer thanits binding partner, e.g., by up to 25 bases, or up to 15 bases, or upto 10 bases. Oligonucleotide sequences and their complements can begenerated by techniques known in the art for generating pairs ofcomplementary oligonucleotides with similar binding energies (or meltingtemperatures) and low inter-pair cross-reactivity (e.g., commercial orpublic software for selecting probes or primers for multiplexed nucleicacid assays). Oligonucleotide sequences can include naturally occurringnucleic acid bases as well as non-naturally occurring and/or modifiedbases. For example, the oligonucleotide sequences can include Iso-dCand/or Iso-dG, which are chemical variants of cytosine and guanine,respectively, available from EraGen Biosciences, Inc. (www.eragen.com).Incorporation of such modified bases into oligonucleotide sequenceseffectively expands the genetic alphabet and permits synthesis ofoligonucleotides that have increased specificity and decreased mismatchhybridization potential. For example, an oligonucleotide containingIso-dC can be designed so that it will hybridize to a complementaryoligo containing Iso-dG but will not hybridize to any naturallyoccurring nucleic acids sequence. In addition or alternatively,oligonucleotide sequences and their complements can include dimerizedand/or dendritic oligonucleotide sequences. Moreover, theoligonucleotide sequences and their complements can be multi-functional,e.g., including (a) a first segment designed to bind to a capturereagent via a first segment complement (i.e., the capture reagentincludes a targeting agent complement that is complementary to the firstsegment of the multi-functional targeting agent), and (b) a secondsegment designed to bind to an additional moiety present in the assaymedium. In this regard, reference is made to copending applicationserial no. PCT/US15/30925, filed May 15, 2015.

In one approach, a computer algorithm can be used to generateoligonucleotide sequence pairs based on one or more, and preferably all,of the following rules: (i) GC content between about 40-60%, e.g.,40-50%; (ii) a maximum string of base repeats in a sequence of no morethan three; (iii) a maximum number of base pair matches of six betweensequences in different pairs, with no more than four matches in a row;(iv) a rejection of sequences with predicted hairpin loop sizes between2-5, oligonucleotides if they have four or more base-pair matches in thestem region (loop sizes of six or greater are retained); and (v) ahigher free energy (ΔG°) of the specific interactions resulting from a40-60% or 40-50% GC content (ΔG° is dependent on temperature and saltconcentration, and at 23° C. and 200 mM of a monovalent cation, pH 7.0,ΔG° preferably exceeds −15 kcal/mol). In a particular embodiment, atleast relative GC content and ΔG° are considered, along with one or morethe rules identified above, in oligonucleotide selection. In oneembodiment, it may be advantageous to design oligonucleotide sequencesthat minimize non-specific binding and this can be achieved by a varietyof methods. For example, one can design oligonucleotide/oligonucleotidepairs that form no more than three consecutive G/C pairs with otheroligonucleotides used in the assay. Alternatively or additionally, oneor more of the following configurations can be avoided: formation ofsingle nucleotide loops or single nucleotide mismatches positionedbetween G/C-rich sequences when paired with other oligonucleotides usedin the assay.

In one embodiment, the targeting agent and the targeting agentcomplement comprise a pair of oligonucleotides, wherein the pair isselected from one of the following sequence pairs in Table 1(a):

pair # Sequence (5′-3′) 1 Acatcggtagtt (SEQ ID NO: 1)Aactaccgatgt (SEQ ID NO: 2) 2 acgtcccagttg (SEQ ID NO: 3)caactgggacgt (SEQ ID NO: 4) 3 agaagaagatcc (SEQ ID NO: 5)ggatcttcttct (SEQ ID NO: 6) 4 aggttcagtgca (SEQ ID NO: 7)tgcactgaacct (SEQ ID NO: 8) 5 atcaggatacgc (SEQ ID NO: 9)gcgtatcctgat (SEQ ID NO: 10) 6 atcattaccacc (SEQ ID NO: 11)ggtggtaatgat (SEQ ID NO: 12) 7 attaacgggagc (SEQ ID NO: 13)gctcccgttaat (SEQ ID NO: 14) 8 cagaggtcttaa (SEQ ID NO: 15)ttaagacctctg (SEQ ID NO: 16) 9 caggtgtccatt (SEQ ID NO: 17)aatggacacctg (SEQ ID NO: 18) 10 catccaatccag (SEQ ID NO: 19)ctggattggatg (SEQ ID NO: 20) 11 cctacgatatac (SEQ ID NO: 21)gtatatcgtagg (SEQ ID NO: 22) 12 cgaatgtagagt (SEQ ID NO: 23)actctacattcg (SEQ ID NO: 24) 13 cggtttgagata (SEQ ID NO: 25)tatctcaaaccg (SEQ ID NO: 26) 14 cttacaacgcca (SEQ ID NO: 27)tggcgttgtaag (SEQ ID NO: 28) 15 ctttctcggcac (SEQ ID NO: 29)gtgccgagaaag (SEQ ID NO: 30) 16 gacataaagcga (SEQ ID NO: 31)tcgctttatgtc (SEQ ID NO: 32) 17 gccatagtctct (SEQ ID NO: 33)agagactatggc (SEQ ID NO: 34) 18 gctaattcacca (SEQ ID NO: 35)tggtgaattagc (SEQ ID NO: 36) 19 ggtcgtgtttca (SEQ ID NO: 37)tgaaacacgacc (SEQ ID NO: 38) 20 gttgattctgtc (SEQ ID NO: 39)gacagaatcaac (SEQ ID NO: 40) 21 tacccggaataa (SEQ ID NO: 41)ttattccgggta (SEQ ID NO: 42) 22 tgcttgacttgg (SEQ ID NO: 43)ccaagtcaagca (SEQ ID NO: 44) 23 ttccacttaggg (SEQ ID NO: 45)ccctaagtggaa (SEQ ID NO: 46) 24 ttgtctagcggc (SEQ ID NO: 47)gccgctagacaa (SEQ ID NO: 48) 25 tttcccttgcta (SEQ ID NO: 49)tagcaagggaaa (SEQ ID NO: 50)

In a particular embodiment, the targeting agent and the targeting agentcomplement comprise a pair of oligonucleotides, wherein the pair isselected from one of the following sequence pairs in Table 1(b):

Pair Name Modification Sequence 1 3′ Thiol Oligo 12b-1 3′-thiol C3 SSacatcggtagtt (SEQ ID NO: 1) 3′ Biotin Oligo 12b-1 3′ biotinaactaccgatgt (SEQ ID NO: 2) 3 3′ Thiol Oligo 12b-5 3′-thiol C3 SSagaagaagatcc (SEQ ID NO: 5) 3′ Biotin Oligo 12b-5 3′ biotinggatcttcttct (SEQ ID NO: 6) 6 3′ Thiol Oligo 12b-12 3′-thiol C3 SSatcattaccacc (SEQ ID NO: 11) 3′ Biotin Oligo 12b- 3′ biotinggtggtaatgat (SEQ ID NO: 12) 12 7 3′ Thiol Oligo 12b-14 3′-thiol C3 SSattaacgggagc (SEQ ID NO: 13) 3′ Biotin Oligo 12b- 3′ biotingctcccgttaat (SEQ ID NO: 14) 14 8 3′ Thiol Oligo 12b-17 3′-thiol C3 SScagaggtcttaa (SEQ ID NO: 15) 3′ Biotin Oligo 12b- 3′ biotinttaagacctctg (SEQ ID NO: 16) 17 9 3′ Thiol Oligo 12b-18 3′-thiol C3 SScaggtgtccatt (SEQ ID NO: 17) 3′ Biotin Oligo 12b- 3′ biotinaatggacacctg (SEQ ID NO: 18) 18 11 3′ Thiol Oligo 12b-20 3′-thiol C3 SScctacgatatac (SEQ ID NO: 21) 3′ Biotin Oligo 12b- 3′ biotingtatatcgtagg (SEQ ID NO: 22) 20 12 3′ Thiol Oligo 12b-21 3′-thiol C3 SScgaatgtagagt (SEQ ID NO: 23) 3′ Biotin Oligo 12b- 3′ biotinactctacattcg (SEQ ID NO: 24) 21 13 3′ Thiol Oligo 12b-22 3′-thiol C3 SScggtttgagata (SEQ ID NO: 25) 3′ Biotin Oligo 12b- 3′ biotintatctcaaaccg (SEQ ID NO: 26) 22 16 3′ Thiol Oligo 12b-26 3′-thiol C3 SSgacataaagcga (SEQ ID NO: 31) 3′ Biotin Oligo 12b- 3′ biotintcgctttatgtc (SEQ ID NO: 32) 26 17 3′ Thiol Oligo 12b-28 3′-thiol C3 SSgccatagtctct (SEQ ID NO: 33) 3′ Biotin Oligo 12b- 3′ biotinagagactatggc (SEQ ID NO: 34) 28 18 3′ Thiol Oligo 12b-30 3′-thiol C3 SSgctaattcacca (SEQ ID NO: 35) 3′ Biotin Oligo 12b- 3′ biotintggtgaattagc (SEQ ID NO: 36) 30 20 3′ Thiol Oligo 12b-33 3′-thiol C3 SSgttgattctgtc (SEQ ID NO: 39) 3′ Biotin Oligo 12b- 3′ biotingacagaatcaac (SEQ ID NO: 40) 33 23 3′ Thiol Oligo 12b-41 3′-thiol C3 SSttccacttaggg (SEQ ID NO: 45) 3′ Biotin Oligo 12b- 3′ biotinccctaagtggaa (SEQ ID NO: 46) 41 25 3′ Thiol Oligo 12b-43 3′-thiol C3 SStttcccttgcta (SEQ ID NO: 49) 3′ Biotin Oligo 12b- 3′ biotintagcaagggaaa (SEQ ID NO: 50) 43

For example, the invention includes one of the sets of ten pair oftargeting agent and the targeting agent complement shown in Table 1(c):

3′ Thiol Oligo Corresponding 3′ Biotinylated Oligo Set (1)5′-ATC ATT ACC ACC/3ThioMC3-D/-3′ 5′-GGT GGT AAT GAT/3Bio/-3′(SEQ ID NO: 11) (SEQ ID NO: 12) 5′-CCT ACG ATA TAC/3ThioMC3-D/-3′5′-GTA TAT CGT AGG/3Bio/-3′ (SEQ ID NO: 21) (SEQ ID NO: 22)5′-CGG TTT GAG ATA/3ThioMC3-D/-3′ 5′-TAT CTC AAA CCG/3Bio/-3′(SEQ ID NO: 25) (SEQ ID NO: 26) 5′-GAC ATA AAG CGA/3ThioMC3-D/-3′5′-TCG CTT TAT GTC/3Bio/-3′ (SEQ ID NO: 31) (SEQ ID NO: 32)5′-GTT GAT TCT GTC/3ThioMC3-D/-3′ 5′-GAC AGA ATC AAC/3Bio/-3′(SEQ ID NO: 39) (SEQ ID NO: 40) 5′-GCT AAT TCA CCA/3ThioMC3-D/-3′5′-TGG TGA ATT AGC/3Bio/-3′ (SEQ ID NO: 35) (SEQ ID NO: 36)5′-TTT CCC TTG CTA/3ThioMC3-D/-3′ 5′-TAG CAA GGG AAA/3Bio/-3′(SEQ ID NO: 49) (SEQ ID NO: 50) 5′-AGA AGA AGA TCC/3ThioMC3-D/-3′5′-GGA TCT TCT TCT/3Bio/-3′ (SEQ ID NO: 5) (SEQ ID NO: 6)5′-ACA TCG GTA GTT/3ThioMC3-D/-3′ 5′-AAC TAC CGA TGT/3Bio/-3′(SEQ ID NO: 1) (SEQ ID NO: 2 5′-GCC ATA GTC TCT/3ThioMC3-D/-3′5′-AGA GAC TAT GGC/3Bio/-3′ (SEQ ID NO: 33) (SEQ ID NO: 34) Set (2)5′-CGA ATG TAG AGT/3ThioMC3-D/-3′ 5′-ACT CTA CAT TCG/3Bio/-3′(SEQ ID NO: 23) (SEQ ID NO: 24) 5′-TTC CAC TTA GGG/3ThioMC3-D/-3′5′-CCC TAA GTG GAA/3Bio/-3′ (SEQ ID NO: 45) (SEQ ID NO: 46)5′-CAG AGG TCT TAA/3ThioMC3-D/-3′ 5′-TTA AGA CCT CTG/3Bio/-3′(SEQ ID NO: 15) (SEQ ID NO: 16) 5′-ACG TCC CAG TTG/3ThioMC3-D/-3′5′-CAA CTG GGA CGT/3Bio/-3′ (SEQ ID NO: 3) (SEQ ID NO: 4)5′-AGG TTC AGT GCA/3ThioMC3-D/-3′ 5′-TGC ACT GAA CCT/3Bio/-3′(SEQ ID NO: 7) (SEQ ID NO: 8) 5′-ATC AGG ATA CGC/3ThioMC3-D/-3′5′-GCG TAT CCT GAT/3Bio/-3′ (SEQ ID NO: 9) (SEQ ID NO: 10)5′-CAT CCA ATC CAG/3ThioMC3-D/-3′ 5′-CTG GAT TGG ATG/3Bio/-3′(SEQ ID NO: 19) (SEQ ID NO: 20) 5′-CTT ACA ACG CCA/3ThioMC3-D/-3′5′-TGG CGT TGT AAG/3Bio/-3′ (SEQ ID NO: 27) (SEQ ID NO: 28)5′-CTT TCT CGG CAC/3ThioMC3-D/-3′ 5′-GTG CCG AGA AAG/3Bio/-3′(SEQ ID NO: 29) (SEQ ID NO: 30) 5′-GGT CGT GTT TCA/3ThioMC3-D/-3′5′-TGA AAC ACG ACC/3Bio/-3′ (SEQ ID NO: 37) (SEQ ID NO: 38) Set (3)5′-ATT AAC GGG AGC/3ThioMC3-D/-3′ 5′-GCT CCC GTT AAT/3Bio/-3′(SEQ ID NO: 13) (SEQ ID NO: 14) 5′-CAG GTG TCC ATT/3ThioMC3-D/-3′5′-AAT GGA CAC CTG/3Bio/-3′ (SEQ ID NO: 17) (SEQ ID NO: 18)5′-CGA ATG TAG AGT/3ThioMC3-D/-3′ 5′-ACT CTA CAT TCG/3Bio/-3′(SEQ ID NO: 23) (SEQ ID NO: 24) 5′-TTC CAC TTA GGG/3ThioMC3-D/-3′5′-CCC TAA GTG GAA/3Bio/-3′ (SEQ ID NO: 45) (SEQ ID NO: 46)5′-CAG AGG TCT TAA/3ThioMC3-D/-3′ 5′-TTA AGA CCT CTG/3Bio/-3′(SEQ ID NO: 15) (SEQ ID NO: 16) 5′-ACG TCC CAG TTG/3ThioMC3-D/-3′5′-CAA CTG GGA CGT/3Bio/-3′ (SEQ ID NO: 3) (SEQ ID NO: 4)5′-AGG TTC AGT GCA/3ThioMC3-D/-3′ 5′-TGC ACT GAA CCT/3Bio/-3′(SEQ ID NO: 7) (SEQ ID NO: 8) 5′-ATC AGG ATA CGC/3ThioMC3-D/-3′5′-GCG TAT CCT GAT/3Bio/-3′ (SEQ ID NO: 9) (SEQ ID NO: 10)5′-CAT CCA ATC CAG/3ThioMC3-D/-3′ 5′-CTG GAT TGG ATG/3Bio/-3′(SEQ ID NO: 19) (SEQ ID NO: 20) 5′-CTT ACA ACG CCA/3ThioMC3-D/-3′5′-TGG CGT TGT AAG/3Bio/-3′ (SEQ ID NO: 27) (SEQ ID NO: 28) Set (4)5′-GCT AAT TCA CCA/3ThioMC3-D/-3′ 5′-TGG TGA ATT AGC/3Bio/-3′(SEQ ID NO: 35) (SEQ ID NO: 36) 5′-TTT CCC TTG CTA/3ThioMC3-D/-3′5′-TAG CAA GGG AAA/3Bio/-3′ (SEQ ID NO: 49) (SEQ ID NO: 50)5′-AGA AGA AGA TCC/3ThioMC3-D/-3′ 5′-GGA TCT TCT TCT/3Bio/-3′(SEQ ID NO: 5) (SEQ ID NO: 6) 5′-ACA TCG GTA GTT/3ThioMC3-D/-3′5′-AAC TAC CGA TGT/3Bio/-3′ (SEQ ID NO: 1) (SEQ ID NO: 25′-GCC ATA GTC TCT/3ThioMC3-D/-3′ 5′-AGA GAC TAT GGC/3Bio/-3′(SEQ ID NO: 33) (SEQ ID NO: 34) 5′-ATT AAC GGG AGC/3ThioMC3-D/-3′5′-GCT CCC GTT AAT/3Bio/-3′ (SEQ ID NO: 13) (SEQ ID NO: 14)5′-CAG GTG TCC ATT/3ThioMC3-D/-3′ 5′-AAT GGA CAC CTG/3Bio/-3′(SEQ ID NO: 17) (SEQ ID NO: 18) 5′-CGA ATG TAG AGT/3ThioMC3-D/-3′5′-ACT CTA CAT TCG/3Bio/-3′ (SEQ ID NO: 23) (SEQ ID NO: 24)5′-TTC CAC TTA GGG/3ThioMC3-D/-3′ 5′-CCC TAA GTG GAA/3Bio/-3′(SEQ ID NO: 45) (SEQ ID NO: 46) 5′-CAG AGG TCT TAA/3ThioMC3-D/-3′5′-TTA AGA CCT CTG/3Bio/-3′ (SEQ ID NO: 15) (SEQ ID NO: 16) Set (5)5′-ATC ATT ACC ACC/3ThioMC3-D/-3′ 5′-GGT GGT AAT GAT/3Bio/-3′(SEQ ID NO: 11) (SEQ ID NO: 12) 5′-CCT ACG ATA TAC/3ThioMC3-D/-3′5′-GTA TAT CGT AGG/3Bio/-3′ (SEQ ID NO: 21) (SEQ ID NO: 22)5′-CGG TTT GAG ATA/3ThioMC3-D/-3′ 5′-TAT CTC AAA CCG/3Bio/-3′(SEQ ID NO: 25) (SEQ ID NO: 26) 5′-GAC ATA AAG CGA/3ThioMC3-D/-3′5′-TCG CTT TAT GTC/3Bio/-3′ (SEQ ID NO: 31) (SEQ ID NO: 32)5′-GTT GAT TCT GTC/3ThioMC3-D/-3′ 5′-GAC AGA ATC AAC/3Bio/-3′(SEQ ID NO: 39) (SEQ ID NO: 40) 5′-ATC AGG ATA CGC/3ThioMC3-D/-3′5′-GCG TAT CCT GAT/3Bio/-3′ (SEQ ID NO: 9) (SEQ ID NO: 10)5′-CAT CCA ATC CAG/3ThioMC3-D/-3′ 5′-CTG GAT TGG ATG/3Bio/-3′(SEQ ID NO: 19) (SEQ ID NO: 20) 5′-CTT ACA ACG CCA/3ThioMC3-D/-3′5′-TGG CGT TGT AAG/3Bio/-3′ (SEQ ID NO: 27) (SEQ ID NO: 28)5′-CTT TCT CGG CAC/3ThioMC3-D/-3′ 5′-GTG CCG AGA AAG/3Bio/-3′(SEQ ID NO: 29) (SEQ ID NO: 30) 5′-GGT CGT GTT TCA/3ThioMC3-D/-3′5′-TGA AAC ACG ACC/3Bio/-3′ (SEQ ID NO: 37) (SEQ ID NO: 38) Set (6)5′-ATC ATT ACC ACC/3ThioMC3-D/-3′ 5′-GGT GGT AAT GAT/3Bio/-3′(SEQ ID NO: 11) (SEQ ID NO: 12) 5′-CCT ACG ATA TAC/3ThioMC3-D/-3′5′-GTA TAT CGT AGG/3Bio/-3′ (SEQ ID NO: 21) (SEQ ID NO: 22)5′-GAC ATA AAG CGA/3ThioMC3-D/-3′ 5′-TCG CTT TAT GTC/3Bio/-3′(SEQ ID NO: 31) (SEQ ID NO: 32) 5′-GTT GAT TCT GTC/3ThioMC3-D/-3′5′-GAC AGA ATC AAC/3Bio/-3′ (SEQ ID NO: 39) (SEQ ID NO: 40)5′-AGA AGA AGA TCC/3ThioMC3-D/-3′ 5′-GGA TCT TCT TCT/3Bio/-3′(SEQ ID NO: 5) (SEQ ID NO: 6) 5′-ACA TCG GTA GTT/3ThioMC3-D/-3′5′-AAC TAC CGA TGT/3Bio/-3′ (SEQ ID NO: 1) (SEQ ID NO: 25′-CAG GTG TCC ATT/3ThioMC3-D/-3′ 5′-AAT GGA CAC CTG/3Bio/-3′(SEQ ID NO: 17) (SEQ ID NO: 18) 5′-CGA ATG TAG AGT/3ThioMC3-D/-3′5′-ACT CTA CAT TCG/3Bio/-3′ (SEQ ID NO: 23) (SEQ ID NO: 24)5′-ACG TCC CAG TTG/3ThioMC3-D/-3′ 5′-CAA CTG GGA CGT/3Bio/-3′(SEQ ID NO: 3) (SEQ ID NO: 4) 5′-AGG TTC AGT GCA/3ThioMC3-D/-3′5′-TGC ACT GAA CCT/3Bio/-3′ (SEQ ID NO: 7) (SEQ ID NO: 8) Set (7)5′-CGG TTT GAG ATA/3ThioMC3-D/-3′ 5′-TAT CTC AAA CCG/3Bio/-3′(SEQ ID NO: 25) (SEQ ID NO: 26) 5′-GAC ATA AAG CGA/3ThioMC3-D/-3′5′-TCG CTT TAT GTC/3Bio/-3′ (SEQ ID NO: 31) (SEQ ID NO: 32)5′-TTT CCC TTG CTA/3ThioMC3-D/-3′ 5′-TAG CAA GGG AAA/3Bio/-3′(SEQ ID NO: 49) (SEQ ID NO: 50) 5′-AGA AGA AGA TCC/3ThioMC3-D/-3′5′-GGA TCT TCT TCT/3Bio/-3′ (SEQ ID NO: 5) (SEQ ID NO: 6)5′-ATT AAC GGG AGC/3ThioMC3-D/-3′ 5′-GCT CCC GTT AAT/3Bio/-3′(SEQ ID NO: 13) (SEQ ID NO: 14) 5′-CAG GTG TCC ATT/3ThioMC3-D/-3′5′-AAT GGA CAC CTG/3Bio/-3′ (SEQ ID NO: 17) (SEQ ID NO: 18)5′-TTC CAC TTA GGG/3ThioMC3-D/-3′ 5′-CCC TAA GTG GAA/3Bio/-3′(SEQ ID NO: 45) (SEQ ID NO: 46) 5′-CAG AGG TCT TAA/3ThioMC3-D/-3′5′-TTA AGA CCT CTG/3Bio/-3′ (SEQ ID NO: 15) (SEQ ID NO: 16)5′-ATC AGG ATA CGC/3ThioMC3-D/-3′ 5′-GCG TAT CCT GAT/3Bio/-3′(SEQ ID NO: 9) (SEQ ID NO: 10) 5′-CAT CCA ATC CAG/3ThioMC3-D/-3′5′-CTG GAT TGG ATG/3Bio/-3′ (SEQ ID NO: 19) (SEQ ID NO: 20) Set (8)5′-GCT AAT TCA CCA/3ThioMC3-D/-3′ 5′-TGG TGA ATT AGC/3Bio/-3′(SEQ ID NO: 35) (SEQ ID NO: 36) 5′-TTT CCC TTG CTA/3ThioMC3-D/-3′5′-TAG CAA GGG AAA/3Bio/-3′ (SEQ ID NO: 49) (SEQ ID NO: 50)5′-AGA AGA AGA TCC/3ThioMC3-D/-3′ 5′-GGA TCT TCT TCT/3Bio/-3′(SEQ ID NO: 5) (SEQ ID NO: 6) 5′-ACA TCG GTA GTT/3ThioMC3-D/-3′5′-AAC TAC CGA TGT/3Bio/-3′ (SEQ ID NO: 1) (SEQ ID NO: 25′-GCC ATA GTC TCT/3ThioMC3-D/-3′ 5′-AGA GAC TAT GGC/3Bio/-3′(SEQ ID NO: 33) (SEQ ID NO: 34) 5′-ACG TCC CAG TTG/3ThioMC3-D/-3′5′-CAA CTG GGA CGT/3Bio/-3′ (SEQ ID NO: 3) (SEQ ID NO: 4)5′-AGG TTC AGT GCA/3ThioMC3-D/-3′ 5′-TGC ACT GAA CCT/3Bio/-3′(SEQ ID NO: 7) (SEQ ID NO: 8) 5′-ATC AGG ATA CGC/3ThioMC3-D/-3′5′-GCG TAT CCT GAT/3Bio/-3′ (SEQ ID NO: 9) (SEQ ID NO: 10)5′-CAT CCA ATC CAG/3ThioMC3-D/-3′ 5′-CTG GAT TGG ATG/3Bio/-3′(SEQ ID NO: 19) (SEQ ID NO: 20) 5′-CTT ACA ACG CCA/3ThioMC3-D/-3′5′-TGG CGT TGT AAG/3Bio/-3′ (SEQ ID NO: 27) (SEQ ID NO: 28) Set (9)5′-ATT AAC GGG AGC/3ThioMC3-D/-3′ 5′-GCT CCC GTT AAT/3Bio/-3′(SEQ ID NO: 13) (SEQ ID NO: 14) 5′-CAG GTG TCC ATT/3ThioMC3-D/-3′5′-AAT GGA CAC CTG/3Bio/-3′ (SEQ ID NO: 17) (SEQ ID NO: 18)5′-CGA ATG TAG AGT/3ThioMC3-D/-3′ 5′-ACT CTA CAT TCG/3Bio/-3′(SEQ ID NO: 23) (SEQ ID NO: 24) 5′-TTC CAC TTA GGG/3ThioMC3-D/-3′5′-CCC TAA GTG GAA/3Bio/-3′ (SEQ ID NO: 45) (SEQ ID NO: 46)5′-CAG AGG TCT TAA/3ThioMC3-D/-3′ 5′-TTA AGA CCT CTG/3Bio/-3′(SEQ ID NO: 15) (SEQ ID NO: 16) 5′-ATC AGG ATA CGC/3ThioMC3-D/-3′5′-GCG TAT CCT GAT/3Bio/-3′ (SEQ ID NO: 9) (SEQ ID NO: 10)5′-CAT CCA ATC CAG/3ThioMC3-D/-3′ 5′-CTG GAT TGG ATG/3Bio/-3′(SEQ ID NO: 19) (SEQ ID NO: 20) 5′-CTT ACA ACG CCA/3ThioMC3-D/-3′5′-TGG CGT TGT AAG/3Bio/-3′ (SEQ ID NO: 27) (SEQ ID NO: 28)5′-CTT TCT CGG CAC/3ThioMC3-D/-3′ 5′-GTG CCG AGA AAG/3Bio/-3′(SEQ ID NO: 29) (SEQ ID NO: 30) 5′-GGT CGT GTT TCA/3ThioMC3-D/-3′5′-TGA AAC ACG ACC/3Bio/-3′ (SEQ ID NO: 37) (SEQ ID NO: 38) Set (10)5′-GAC ATA AAG CGA/3ThioMC3-D/-3′ 5′-TCG CTT TAT GTC/3Bio/-3′(SEQ ID NO: 31) (SEQ ID NO: 32) 5′-GTT GAT TCT GTC/3ThioMC3-D/-3′5′-GAC AGA ATC AAC/3Bio/-3′ (SEQ ID NO: 39) (SEQ ID NO: 40)5′-GCT AAT TCA CCA/3ThioMC3-D/-3′ 5′-TGG TGA ATT AGC/3Bio/-3′(SEQ ID NO: 35) (SEQ ID NO: 36) 5′-TTT CCC TTG CTA/3ThioMC3-D/-3′5′-TAG CAA GGG AAA/3Bio/-3′ (SEQ ID NO: 49) (SEQ ID NO: 50)5′-AGA AGA AGA TCC/3ThioMC3-D/-3′ 5′-GGA TCT TCT TCT/3Bio/-3′(SEQ ID NO: 5) (SEQ ID NO: 6) 5′-TTC CAC TTA GGG/3ThioMC3-D/-3′5′-CCC TAA GTG GAA/3Bio/-3′ (SEQ ID NO: 45) (SEQ ID NO: 46)5′-CAG AGG TCT TAA/3ThioMC3-D/-3′ 5′-TTA AGA CCT CTG/3Bio/-3′(SEQ ID NO: 15) (SEQ ID NO: 16) 5′-ACG TCC CAG TTG/3ThioMC3-D/-3′5′-CAA CTG GGA CGT/3Bio/-3′ (SEQ ID NO: 3) (SEQ ID NO: 4)5′-AGG TTC AGT GCA/3ThioMC3-D/-3′ 5′-TGC ACT GAA CCT/3Bio/-3′(SEQ ID NO: 7) (SEQ ID NO: 8) 5′-ATC AGG ATA CGC/3ThioMC3-D/-3′5′-GCG TAT CCT GAT/3Bio/-3′ (SEQ ID NO: 9) (SEQ ID NO: 10)

In a specific embodiment, the set comprises set 1 listed in Table 1(c).Alternatively, the set comprises set 2 from Table 1(c); the set can alsocomprise set 3 from Table 1(c); the set further comprises set 4 fromTable 1(c); the set also includes set 5 from Table 1(c); the setincludes set 6 from Table 1(c); the set further comprises set 7 fromTable 1(c); the set can also include set 8 from Table 1(c); the setincludes set 9 from Table 1(c); and/or the set includes set 10 fromTable 1(c).

The targeting agent and targeting agent complement may be present in a1:1 ratio. Alternatively, the targeting agent may be present in anexcess, e.g., in a 2:1 ratio of targeting agent to targeting agentcomplement, to increase the likelihood of binding the targeting agent toits complement.

In one embodiment, suitable linking agents and supplemental linkingagents include chemical moieties that react to form a linking complex.For example, the linking complex is formed by a binding interactionbetween chemical moieties present on the linking agent and supplementallinking agent, e.g., a thiol group and a maleimide or iodoacetamidegroups; an aldehyde and a hydrazide; or an alkyne and an azide.

Alternatively, a linking complex can be formed by a protein-proteinbinding reaction between a linking agent and a supplemental linkingagent. For example, a protein-protein binding reaction can be formed viabinding between a receptor/ligand pair, hapten/antibody pair,antigen/antibody pair, epitope/antibody pair, mimitope/antibody pair,aptamer/target molecule pair, hybridization partners, andintercalater/target molecule pair. In one embodiment, the linking agentis biotin and the supplemental linking agent is streptavidin or avidin(or vice versa); or the linking agent is a peptide and the supplementallinking agent is an anti-peptide antibody (or vice versa).

Certain embodiments described herein employ linking agents that binddirectly to supplemental linking agents. In these and other suchembodiments, the linking and supplemental linking agents that bind toeach other can be replaced with linking and supplemental linking agentsthat can concurrently bind to a bridging agent. In these alternateembodiments, a bridging agent is included in the mixture of the linkingagent and supplemental linking agent, when forming the targetingcomplex. A bridging agent, if one is used, binds to the linking agentand the supplemental linking agent. For example, the bridging agentincludes a binding site for the linking agent and an additional bindingsite for the supplemental linking agent, and the combination of thethree components, i.e., the bridging agent, the linking agent, and thesupplemental linking agent, comprises a bridging complex. In oneembodiment, the bridging agent is streptavidin or avidin (each of whichare tetramers with four independent binding sites for biotin) and thelinking agent and supplemental linking agent are each biotin, such thatthe bridging complex comprises (biotin-(streptavidin (oravidin))-biotin).

In a preferred embodiment, a linking complex is formed by a bindingreaction between a linking agent, L_(A), and a supplemental linkingagent, L_(A)′. A multiplex assay format may be configured to detectanalytes A, B, and C, and therefore, the reagent complexes designed tointeract with those individual analytes may include linking agents andsupplemental linking agents selected from L_(A) and L_(A)′, L_(B) andL_(B)′, and L_(C) and L_(C)′. Each of the linking agent/supplementallinking agent pairs used to construct the binding complexes may be thesame or different. In a preferred embodiment, each of the linkingagent/supplemental linking agent pairs comprise the same set ofreagents. In a particularly preferred embodiment, each of the linkingagent/supplemental linking agent pairs comprise, e.g., a biotin moleculeas the linking agent on a binding reagent and a streptavidin or avidinmolecule on a targeting agent as the supplemental linking agent (or viceversa). In addition, a linking complex can also be formed by a bindingreaction between a biotin molecule on a binding reagent and astreptavidin or avidin molecule on a targeting agent, wherein thestreptavidin or avidin molecule is bound to the targeting agent via areaction with a biotin molecule (acting as a bridging agent) on thetargeting agent. In a preferred embodiment, once a linking complex isformed between a biotin and streptavidin molecule on a binding reagentand a targeting agent, respectively (or vice versa), excess free biotinmolecule can be added to prevent cross-reactivity between additionalbinding reagents and targeting agents that may be combined in solution.

(iv) Solid Phases

A wide variety of solid phases are suitable for use in the methods ofthe present invention including conventional solid phases from the artof binding assays. Solid phases may be made from a variety of differentmaterials including polymers (e.g., polystyrene and polypropylene),ceramics, glass, composite materials (e.g., carbon-polymer compositessuch as carbon-based inks). Suitable solid phases include the surfacesof macroscopic objects such as an interior surface of an assay container(e.g., test tubes, cuvettes, flow cells, cartridges, wells in amulti-well plate, etc.), slides, assay chips (such as those used in geneor protein chip measurements), pins or probes, beads, filtration media,lateral flow media (for example, filtration membranes used in lateralflow test strips), etc.

Suitable solid phases also include particles (including but not limitedto colloids or beads) commonly used in other types of particle-basedassays e.g., magnetic, polypropylene, and latex particles, materialstypically used in solid-phase synthesis e.g., polystyrene andpolyacrylamide particles, and materials typically used inchromatographic applications e.g., silica, alumina, polyacrylamide,polystyrene. The materials may also be a fiber such as a carbon fibril.Microparticles may be inanimate or alternatively, may include animatebiological entities such as cells, viruses, bacterium and the like. Aparticle used in the present method may be comprised of any materialsuitable for attachment to one or more binding reagents, and that may becollected via, e.g., centrifugation, gravity, filtration or magneticcollection. A wide variety of different types of particles that may beattached to binding reagents are sold commercially for use in bindingassays. These include non-magnetic particles as well as particlescomprising magnetizable materials which allow the particles to becollected with a magnetic field. In one embodiment, the particles arecomprised of a conductive and/or semiconductive material, e.g.,colloidal gold particles. The microparticles may have a wide variety ofsizes and shapes. By way of example and not limitation, microparticlesmay be between 5 nanometers and 100 micrometers. Preferablymicroparticles have sizes between 20 nm and 10 micrometers. Theparticles may be spherical, oblong, rod-like, etc., or they may beirregular in shape.

FIG. 6(a)-(c) illustrates various set of particles that can be used inthe present invention. FIG. 6(a) shows a set of particles, provided inone or more vials, containers, or compartments, that are each modifiedwith a distinct targeting agent. Alternatively, as shown in FIG. 6(b), aset of particles can be provided in one or more vials, containers, orcompartments, that are each modified with a distinct binding reagent,the binding reagent being attached to the particle in a targetingcomplex that comprises the binding reagent (through linking asupplemental linking agents) to a targeting agent complement that isbound to a targeting agent on the surface of the particle. Stillfurther, FIG. 6(c) shows yet another embodiment in which a mixed set ofparticles is provided, wherein a subset includes particles that are eachmodified with a distinct binding reagent (as in FIG. 6(b) and a subsetincludes particles that are each modified with a distinct targetingagent (as in FIG. 6(a)). The particles shown in FIG. 6(c) include asubset of preconfigured particles with binding reagents for apre-determined set of analytes and a subset of non-configured particlesthat can be modified by a user to attach an additional set of bindingreagents for an additional set of analytes, i.e., by binding theparticles to an additional set of targeting complexes comprisingadditional binding reagents. These additional reagents may be providedby the user thereby allowing the user to add a set of user-definedassays to a pre-defined set of assays.

The particles used in the present method may be coded to allow for theidentification of specific particles or subpopulations of particles in amixture of particles. The use of such coded particles has been used toenable multiplexing of assays employing particles as solid phasesupports for binding assays. In one approach, particles are manufacturedto include one or more fluorescent dyes and specific populations ofparticles are identified based on the intensity and/or relativeintensity of fluorescence emissions at one or more wave lengths. Thisapproach has been used in the Luminex xMAP systems (see, e.g., U.S. Pat.No. 6,939,720) and the Becton Dickinson Cytometric Bead Array systems.Alternatively, particles may be coded through differences in otherphysical properties such as size, shape, imbedded optical patterns andthe like. As indicated by the cross-hatching of the particles in FIGS.6(a)-(c), one or more particles provided in a mixture or set ofparticles may be coded to be distinguishable from other particles in themixture by virtue of particle optical properties, size, shape, imbeddedoptical patterns and the like.

Alternatively or additionally, the binding reagents can be bound viabinding reagent complexes to different discrete binding domains on oneor more solid phases, e.g., as in a binding array, such that discreteassay signals are generated on each binding domain and therefore, thedifferent analytes bound to those domains can be measured independently.In one example of such an embodiment, the binding domains are preparedby immobilizing, on one or more surfaces, discrete domains of targetingagents that, through a binding reagent complex built on the individualdomains, are configured to bind analytes of interest. Optionally, thesurface(s) may define, in part, one or more boundaries of a container(e.g., a flow cell, well, cuvette, etc.) which holds the sample orthrough which the sample is passed. In a preferred embodiment,individual binding domains are formed on electrodes for use inelectrochemical or electrochemiluminescence assays. Multiplexedmeasurement of analytes on a surface comprising a plurality of bindingdomains using electrochemiluminescence has been used in the Meso ScaleDiagnostics, LLC, MULTI-ARRAY® and SECTOR® Imager line or products (see,e.g., U.S. Pat. Nos. 7,842,246 and 6,977,722, the disclosures of whichare incorporated herein by reference in their entireties).

FIG. 6(d)-(f) illustrate various alternative plate formats includingdistinct binding domains that can be used in the present invention. FIG.6(d) shows a surface bearing a plurality of binding domains that areeach modified with a distinct targeting agent. Alternatively, as shownin FIG. 6(e), a surface can be provided bearing a plurality of bindingdomains that are each modified with a distinct binding reagent, thebinding reagent being attached to the binding domain in a targetingcomplex that comprises the binding reagent (through linking asupplemental linking agents) to a targeting agent complement that isbound to a targeting agent on the binding domain. Still further, FIG.6(f) shows yet another embodiment in which a mixed set of bindingdomains on a surface is provided, wherein a subset includes bindingdomains that are each modified with a distinct binding reagent (as inFIG. 6e ) and a subset includes binding domains that are each modifiedby a distinct targeting agent (as in FIG. 6(d)). The binding domainsshown in FIG. 6(f) include a subset of preconfigured binding domainswith binding reagents for a pre-determined set of analytes and a subsetof non-configured binding domains that can be modified by a user toattach an additional set of binding reagents for an additional set ofanalytes, i.e., by binding these non-configured binding domains to anadditional set of targeting complexes comprising additional bindingreagents. These additional reagents may be provided by the user, therebyallowing the user to add a set of user-defined assays to a pre-definedset of assays.

Reagents, i.e., targeting agents, can be bound to a surface by knownmethods, e.g., established methods for modifying particles or forforming arrays. One non-limiting example of a method of attaching aprotein or oligonucleotide to a surface is illustrated in FIG. 7. Thismethod usesSulfosuccinimidyl-4-(N-maleimidomethyl)cyclohexane-1-carboxylate(Sulfo-SMCC), a well-established heterobifunctional cross-linking agent.Reaction of the N-hydroxysuccinimide (NHS) group of SMCC with bovineserum albumin (BSA) labels the BSA with thiol-reactive maleimide groups.The maleimide groups are, in turn, reacted with thiol-modifiedoligonucleotides to form BSA-oligonucleotide conjugates that are linkedthrough stable thioether bonds. Arrays of these reagents can be formedby printing patterns of the reagents on surfaces that adsorb or reactwith proteins (such as BSA), thereby generating patterned arrays of theassociated oligonucleotides. In one specific example, arrays are formedby printing arrays of the BSA-oligonucleotide conjugates on graphiticcarbon surfaces, preferably screen printed carbon ink electrodes.Surprisingly, we have found that thiol-modified reagents includingthiol-modified oligonucleotides react irreversibly with graphitic carbonsurfaces including screen-printed carbon ink electrodes (even when notconjugated to a protein such as BSA). Accordingly, thiol-modifiedreagents (including thiol modified oligonucleotides and peptides) can beimmobilized on carbon surfaces by incubating the carbon surfaces with asolution containing the reagents and allowing the reagent toirreversibly react with the surface. Furthermore, arrays of suchthiol-modified reagents (including arrays comprising thiol-modifiedoligonucleotides and/or peptides) can be formed by printing solutionscontaining these reagents on carbon surfaces, incubating the patternedsolutions on the surface and allowing the reagents to irreversibly reactwith the surface.

(v) Assay Devices and Supplementary Reagents

The methods of the present invention may be used in a variety of assaydevices and/or formats. The assay devices may include, e.g., assaymodules, such as assay plates, cartridges, multi-well assay plates,reaction vessels, test tubes, cuvettes, flow cells, assay chips, lateralflow devices, etc., having assay reagents (which may include targetingagents or other binding reagents) added as the assay progresses orpre-loaded in the wells, chambers, or assay regions of the assay module.These devices may employ a variety of assay formats for specific bindingassays, e.g., immunoassay or immunochromatographic assays. Illustrativeassay devices and formats are described herein below. In certainembodiments, the methods of the present invention may employ assayreagents that are stored in a dry state and the assay devices/kits mayfurther comprise or be supplied with desiccant materials for maintainingthe assay reagents in a dry state. The assay devices preloaded with theassay reagents can greatly improve the speed and reduce the complexityof assay measurements while maintaining excellent stability duringstorage. The dried assay reagents may be any assay reagent that can bedried and then reconstituted prior to use in an assay. These include,but are not limited to, binding reagents useful in binding assays,enzymes, enzyme substrates, indicator dyes and other reactive compoundsthat may be used to detect an analyte of interest. The assay reagentsmay also include substances that are not directly involved in themechanism of detection but play an auxiliary role in an assay including,but not limited to, blocking agents, stabilizing agents, detergents,salts, pH buffers, preservatives, etc. Reagents may be present in freeform or supported on solid phases including the surfaces of compartments(e.g., chambers, channels, flow cells, wells, etc.) in the assay modulesor the surfaces of colloids, beads, or other particulate supports.

(vi) Measurement Methods

The methods of the invention can be used with a variety of methods formeasuring the amount of an analyte and, in particular, measuring theamount of an analyte bound to a solid phase. Techniques that may be usedinclude, but are not limited to, techniques known in the art such ascell culture-based assays, binding assays (including agglutinationtests, immunoassays, nucleic acid hybridization assays, etc.), enzymaticassays, colorometric assays, etc. Other suitable techniques will bereadily apparent to one of average skill in the art. Some measurementtechniques allow for measurements to be made by visual inspection,others may require or benefit from the use of an instrument to conductthe measurement.

Methods for measuring the amount of an analyte include label freetechniques, which include but are not limited to i) techniques thatmeasure changes in mass or refractive index at a surface after bindingof an analyte to a surface (e.g., surface acoustic wave techniques,surface plasmon resonance sensors, ellipsometric techniques, etc.), ii)mass spectrometric techniques (including techniques like MALDI, SELDI,etc. that can measure analytes on a surface), iii) chromatographic orelectrophoretic techniques, iv) fluorescence techniques (which may bebased on the inherent fluorescence of an analyte), etc.

Methods for measuring the amount of an analyte also include techniquesthat measure analytes through the detection of labels which may beattached directly or indirectly (e.g., through the use of labeledbinding partners of an analyte) to an analyte. Suitable labels includelabels that can be directly visualized (e.g., particles that may be seenvisually and labels that generate an measurable signal such as lightscattering, optical absorbance, fluorescence, chemiluminescence,electrochemiluminescence, radioactivity, magnetic fields, etc). Labelsthat may be used also include enzymes or other chemically reactivespecies that have a chemical activity that leads to a measurable signalsuch as light scattering, absorbance, fluorescence, etc. The use ofenzymes as labels has been well established in in Enzyme-LinkedImmunoSorbent Assays, also called ELISAs, Enzyme ImmunoAssays or EIAs.In the ELISA format, an unknown amount of antigen is affixed to asurface and then a specific antibody is washed over the surface so thatit can bind to the antigen. This antibody is linked to an enzyme, and inthe final step a substance is added that the enzyme converts to aproduct that provides a change in a detectable signal. The formation ofproduct may be detectable, e.g., due a difference, relative to thesubstrate, in a measurable property such as absorbance, fluorescence,chemiluminescence, light scattering, etc. Certain (but not all)measurement methods that may be used with solid phase binding methodsaccording to the invention may benefit from or require a wash step toremove unbound components (e.g., labels) from the solid phaseAccordingly, the methods of the invention may comprise such a wash step.

In one embodiment, an analyte(s) of interest in the sample may bemeasured using electrochemiluminescence-based assay formats, e.g.electrochemiluminescence (ECL) based immunoassays. The high sensitivity,broad dynamic range and selectivity of ECL are important factors formedical diagnostics. Commercially available ECL instruments havedemonstrated exceptional performance and they have become widely usedfor reasons including their excellent sensitivity, dynamic range,precision, and tolerance of complex sample matrices. Species that can beinduced to emit ECL (ECL-active species) have been used as ECL labels,e.g., i) organometallic compounds where the metal is from, for example,the noble metals of group VIII, including Ru-containing andOs-containing organometallic compounds such as thetris-bipyridyl-ruthenium (RuBpy) moiety and ii) luminol and relatedcompounds. Species that participate with the ECL label in the ECLprocess are referred to herein as ECL coreactants. Commonly usedcoreactants include tertiary amines (e.g., see U.S. Pat. No. 5,846,485),oxalate, and persulfate for ECL from RuBpy and hydrogen peroxide for ECLfrom luminol (see, e.g., U.S. Pat. No. 5,240,863). The light generatedby ECL labels can be used as a reporter signal in diagnostic procedures(Bard et al., U.S. Pat. No. 5,238,808, herein incorporated byreference). For instance, an ECL label can be covalently coupled to abinding agent such as an antibody, nucleic acid probe, receptor orligand; the participation of the binding reagent in a bindinginteraction can be monitored by measuring ECL emitted from the ECLlabel. Alternatively, the ECL signal from an ECL-active compound may beindicative of the chemical environment (see, e.g., U.S. Pat. No.5,641,623 which describes ECL assays that monitor the formation ordestruction of ECL coreactants). For more background on ECL, ECL labels,ECL assays and instrumentation for conducting ECL assays see U.S. Pat.Nos. 5,093,268; 5,147,806; 5,324,457; 5,591,581; 5,597,910; 5,641,623;5,643,713; 5,679,519; 5,705,402; 5,846,485; 5,866,434; 5,786,141;5,731,147; 6,066,448; 6,136,268; 5,776,672; 5,308,754; 5,240,863;6,207,369; 6,214,552 and 5,589,136 and Published PCT Nos. W099/63347;WOOO/03233; W099/58962; W099/32662; W099/14599; W098/12539; W097/36931and W098/57154, all of which are incorporated herein by reference.

The methods of the invention may be applied to singleplex or multiplexformats where multiple assay measurements are performed on a singlesample. Multiplex measurements that can be used with the inventioninclude, but are not limited to, multiplex measurements i) that involvethe use of multiple sensors; ii) that use discrete assay domains on asurface (e.g., an array) that are distinguishable based on location onthe surface; iii) that involve the use of reagents coated on particlesthat are distinguishable based on a particle property such as size,shape, color, etc.; iv) that produce assay signals that aredistinguishable based on optical properties (e.g., absorbance oremission spectrum) or v) that are based on temporal properties of assaysignal (e.g., time, frequency or phase of a signal).

(vii) Kits

In one embodiment, the invention provides a kit including a surface,e.g., multi-well plate or plurality of particles, comprising a pluralityof discrete binding domains each including a first and second targetingagent, e.g., a first and second oligonucleotide, and, in a separatevial, container, or compartment, a first oligonucleotide complementbound to a linking agent and a second oligonucleotide complement boundto a second linking agent. In a preferred embodiment, (i) the firsttargeting agent and the first targeting agent complement comprise afirst pair of targeting agents, and (ii) the second targeting agent andthe second targeting agent complement comprise a second pair oftargeting agents. Preferably, the first and second pair of targetingagents comprise a first and second pair of oligonucleotides,respectively, selected from the pairs of sequences listed in Table 1.

The kit further includes, in separate vials, containers, orcompartments, at least 4 oligonucleotides comprising a differentsequence selected from the sequences listed in Table 1(a) and/or Table1(b). These sequences may include four sequences selected from differentpairs or may include more than one member of a pair.

The kit can include at least 7, 10, 16, or 25 surface boundoligonucleotides and corresponding oligonucleotide complements. Theoligonucleotides configured for use as targeting agent complements canbe provided pre-bound to a binding reagent such as an antibody or can beprovided modified with a linking agent for attachment to a bindingreagent by the user. Optionally, each oligonucleotide complement in thekit is coupled to a different binding reagent, e.g., antibody. Thesurface-bound oligonucleotides can be incorporated into an arraycomprising a plurality of at least 5 (7, 10, 16, or 25) oligonucleotidesimmobilized to each binding domain such that a different oligonucleotidesequence is immobilized to a discrete binding domain. In a specificembodiment, a multi-well plate can include one or more copies of anoligonucleotide array as described herein within at least one well ofthe plate, wherein the array is positioned on a plurality of bindingdomains. The plate can include at least 24, 96, or 384 wells and thearray can include at least 7 oligonucleotides, or at least 10, 16, or 25oligonucleotides.

In a specific embodiment, the kit includes a multi-well plate having oneor more copies of an oligonucleotide array within at least one well(s)of the plate, the array is positioned on a plurality of binding domains,wherein one or more and optionally, at least 4 of the binding domainshave immobilized thereon a different oligonucleotide sequence selectedfrom a different sequence pair from the set of sequence pairs listed inTable 1(a). In a specific embodiment, the kit includes a multi-wellplate having one or more copies of an oligonucleotide array within atleast one well(s) of the plate, the array is positioned on a pluralityof binding domains, wherein one or more and optionally, at least 4 ofthe binding domains have immobilized thereon a different oligonucleotidesequence selected from a different sequence pair from the set ofsequence pairs listed in Table 1(b). The kit may further comprise anadditional set of oligonucleotides comprised of two or moreoligonucleotides selected from the set of sequences in Table 1(a), andin a specific embodiment Table 1(b), wherein the additionaloligonucleotides are complementary to the immobilized oligonucleotides.

The kit may further include one or more additional containers, vesselsor compartments comprising: (i) a first binding reagent comprising afirst linking agent, wherein the first binding reagent is specific for afirst analyte in the sample, (ii) a first targeting agent complementcomprising a supplemental linking agent, provided in a separatecontainer, vessel or compartment or as a component of (i), (iii) asecond binding reagent comprising a second linking agent, wherein thesecond binding reagent is specific for a second analyte in the sample,and (iv) a second targeting agent complement comprising a secondsupplemental linking agent, provided in a separate container, vessel orcompartment or as a component of (iii).

Alternatively, the invention provides a kit for measuring a plurality ofdifferent analytes in a sample, the kit comprising: (a) a container,vessel or compartment comprising a solid support including a firsttargeting agent immobilized to a first region of the solid support and asecond targeting agent immobilized to a second region of said solidsupport; and (b) one or more additional containers, vessels orcompartments comprising: (i) a first binding reagent comprising a firstlinking agent, wherein said first binding reagent is specific for afirst analyte in said sample, (ii) a first targeting agent complementcomprising a supplemental linking agent, provided in a separatecontainer, vessel or compartment or as a component of (b)(i), (iii) asecond binding reagent comprising a second linking agent, wherein saidsecond binding reagent is specific for a second analyte in said sample,and (iv) a second targeting agent complement comprising a second linkingagent complement, provided in a separate container, vessel orcompartment or as a component of (b)(iii).

The invention also contemplates a kit for measuring a plurality ofdifferent analytes in a sample, the kit comprising: (a) a container,vessel or compartment comprising on a solid support a first targetingagent immobilized to a first region of said solid support and a secondtargeting agent immobilized to a second region of said solid support;and (b) four or more additional containers, vessels or compartmentscomprising: (i) a first container comprising a first binding reagentcomprising a first linking agent, wherein said first binding reagent isspecific for a first analyte in said sample, (ii) a second containercomprising a first targeting agent complement comprising a supplementallinking agent, provided in a separate container, vessel or compartment,(iii) a third container comprising a second binding reagent comprising asecond linking agent, wherein said second binding reagent is specificfor a second analyte in said sample, and (iv) a fourth containercomprising a second targeting agent complement comprising a secondsupplemental linking agent, provided in a separate container, vessel orcompartment.

Still further, the invention provides a kit for measuring a plurality ofdifferent analytes in a sample, the kit comprising: (a) a container,vessel or compartment comprising on a solid support a first targetingagent immobilized to a first region of said solid support and a secondtargeting agent immobilized to a second region of said solid support;and (b) two or more additional containers, vessels or compartmentscomprising: (i) a first container comprising a first binding reagentcomprising a first linking agent, wherein said first binding reagent isspecific for a first analyte in said

sample, and a first targeting agent complement comprising a supplementallinking agent, and (ii) a second container comprising a second bindingreagent comprising a second linking agent, wherein said second bindingreagent is specific for a second analyte in said sample, and a secondtargeting agent complement comprising a second supplemental linkingagent.

In a specific embodiment, the invention provides a kit comprising: (a) amulti-well plate comprising a plurality of discrete binding domains eachcomprising a first and second oligonucleotide, respectively, each ofsaid first and second oligonucleotides are selected from the groupconsisting of the sequences listed in Table 1(a) and/or (b).

The kit can also include instructions for use of the multi-well plate ina method of conducting a binding assay for a plurality of analytes, saidmethod comprising the steps of:

(a) forming a first binding reagent complex comprising a first bindingreagent specific for a first analyte in said plurality of analytes andsaid first oligonucleotide, wherein said first binding reagent is boundto a linking agent and said first oligonucleotide is bound to asupplemental linking agent wherein said first binding reagent complex isformed by a reaction between said linking agent and said supplementallinking agent;

(b) forming a second binding reagent complex comprising a second bindingreagent specific for a second analyte in said plurality of analytes andsaid second oligonucleotide, wherein said second binding reagent isbound to a second linking agent and said second oligonucleotide is boundto a second supplemental linking agent wherein said second bindingreagent complex is formed by a reaction between said second linkingagent and said second supplemental linking agent;

(c) mixing said first and second binding reagent complexes with said twoor more binding domains each linked to a first oligonucleotidecomplement and a second oligonucleotide complement, respectively, underconditions sufficient to bind said first oligonucleotide to said firstoligonucleotide complement and said second oligonucleotide to saidsecond oligonucleotide complement;

(d) mixing a sample comprising said plurality of analytes to the mixtureformed in step (c);

(e) adding a plurality of additional binding reagents to the mixtureformed in step (d), wherein said plurality of additional bindingreagents includes (i) a first detection reagent specific for said firstanalyte and/or a first binding reagent-first analyte complex; and (ii) asecond detection reagent specific for said second analyte and/or asecond binding reagent-second analyte complex; and

(f) measuring the amount of said first and second analytes bound to saidbinding domains.

In one specific embodiment, a multi-well assay plate can be used toconfigure an end-user developed assay panel, i.e., an assay panel builtby the end-user with his/her binding reagents to conduct an assay withthe plate. In this embodiment, the end-user designates which bindingreagent is bound to each binding domain. A multi-well assay plate isprovided that includes a plurality of discrete binding domains includinga first binding domain with a first targeting agent and a second bindingdomain with a second targeting agent and, optionally, additional bindingdomains with additional targeting agents. Each of the binding domainsare functionalized by the user by selecting individual binding reagentsthat will be attached to each of the plurality of binding domains via abinding reagent complex, as described herein. In a separate vial,container, or compartment, a set of targeting reagents (each attached toa linking agent) is provided that includes a first targeting agentcomplement, a second targeting agent complement, and optionallyadditional targeting agent complements. The first targeting agent andfirst targeting agent complement and the second targeting agent andsecond targeting agent complement constitute a first and second pair oftargeting agents, respectively. Similarly, any additional targetingagent complements form pairs with the different additional targetingagents on the binding domains. In one preferred embodiment, thetargeting agents and targeting agent complements are oligonucleotides(i.e., an oligonucleotide and its complement). In this embodiment, thefirst and second pairs of targeting agents, and any additional pairs oftargeting agents, are selected from the list of sequences provided inTable 1(a) and/or Table 1(b).

Therefore, the user selects which targeting agent/targeting agentcomplement will be bound to each specific binding domain. The user alsoselects which binding reagent will be bound to each specific bindingdomain and forms a binding reagent complex that includes the targetingagent complement of the targeting agent attached to the designatedbinding domain.

The kit may provide reagents for the users to attach the supplementarylinking agent to the users' binding reagents. When biotin is thesupplementary linking agent, the kit may include biotin modified with areactive functional group such as an NHS ester or hydrazide ormaleimide. The plate and/or set of targeting reagents can furtherinclude a labeling kit for attaching a detectable label to an assaycomponent, such as a detection reagent. For example, if the multi-wellassay plate is configured to conduct an electrochemiluminescencereaction, the labeling kit can include a SULFO-TAG™ NHS ester, LC-biotinNHS ester, an optional spin column, and optional labeling buffersolution. Further provided can be ECL read buffer and optional assay andantibody diluents.

The set of targeting reagents preferably includes a quantity oftargeting reagents that matches the number of binding domains present inthe multi-well plate. For example, if the multi-well plate includes tendiscrete binding domains, a set of 10 targeting reagents are used withthat multi-well plate.

The targeting agents may be provided with a linking agent that directlybinds to the supplementary linking agent, e.g., streptavidin or avidinwhen the supplementary linking agent is biontin. When the linking agentand supplementary linking agent are configured to be linked through abridging agent (e.g., when both the linking and supplementary linkingagents are biotin), the kit may also provide a bridging reagent solution(e.g., a solution of streptavidin or avidin) that can be used to attachthe binding reagent to the targeting agent complement. The kit may alsoprovide a reaction buffer that provides the appropriate conditions forthe linking/bridging reactions and a reaction stop solution. When one ormore of the linking reagents are biotin, the stop solution may includefree biotin to block any unused biotin-binding sites in streptavidin oravidin that is present as a linking agent, supplemental linking agent orbridging agent.

In this embodiment, the user supplies the binding reagents, e.g.,capture and detection antibodies, and designates which binding reagentwill be attached to each of the binding domains. The binding reagent,e.g., capture antibody, is labeled with a selected linking agent, e.g.,biotin, and attached to a member of a targeting agent pair via asupplemental linking agent, e.g., streptavidin. Meanwhile, the plate isprepared by binding the targeting agent to the selected binding domain.The modified binding reagent contacts the surface to form asurface-bound binding reagent complex that can be used in a subsequentbinding assay for an analyte recognized by the binding reagent. Theanalyte of interest is detected by contacting the binding domain with alabeled binding reagent and measuring the presence of the label presentat that binding domain.

Alternatively, a multi-well assay plate can be configured based on auser's specifications, e.g., from a catalog of available multiplexedassay panels and/or a user can select a set of analytes to configure auser-customized multiplexed assay for that set of analytes. Amultiplexed assay panel should be selected and optimized such thatindividual assays function well together. For example, the sample mayrequire dilution prior to being assayed. Sample dilutions for specificsample matrices of interest are optimized for a given panel to minimizesample matrix effects and to maximize the likelihood that all theanalytes in the panel will be within the dynamic range of the assay. Ina preferred embodiment, all of the analytes in the panel are analyzedwith the same sample dilution in at least one sample type. In anotherpreferred embodiment, all of the analytes in a panel are measured usingthe same dilution for most sample types.

For a given immunoassay panel, the detection antibody concentration andthe number of detectable labels per protein (L/P ratio) for thedetection antibody are adjusted to bring the expected levels of allanalytes into a quantifiable range at the same sample dilution. If onewants to increase the high end of the quantifiable range for a givenanalyte, then the UP can be decreased and/or the detection antibodyconcentration is decreased. On the other hand, if one wants to increasethe lower end of the quantifiable range, the UP can be increased, thedetection antibody concentration can be increased if it is not at thesaturation level, and/or the background signal can be lowered.

Calibration standards for use with an assay panel are selected toprovide the appropriate quantifiable range with the recommended sampledilution for the panel. The calibration standards have knownconcentrations of one of more of the analytes in the panel.Concentrations of the analytes in unknown samples are determined bycomparison to these standards. In one embodiment, calibration standardscomprise mixtures of the different analytes measured by an assay panel.Preferably, the analyte levels in a combined calibrator are selectedsuch that the assay signals for each analyte are comparable, e.g.,within a factor of two, a factor of five or a factor of 10. In anotherembodiment, calibration standards include mixtures of analytes frommultiple different assay panels.

A calibration curve may be fit to the assay signals measured withcalibration standards using, e.g., curve fits known in the art such aslinear fits, 4-parameter logistic (4-PL) and 5-parameter (5-PL) fits.Using such fits, the concentration of analytes in an unknown sample maybe determined by backfitting the measured assay signals to thecalculated fits. Measurements with calibration standards may also beused to determine assay characteristics such as the limit of detection(LOD), limit of quantification (LOQ), dynamic range, and limit oflinearity (LOL).

As part of a multiplexed panel development, assays are optimized toreduce calibrator and detection antibody non-specific binding. Insandwich immunoassays, specificity mainly comes from capture antibodybinding. Some considerations for evaluating multiplexed panels include:(a) detection antibody non-specific binding to capture antibodies isreduced to lower background of assays in the panel, and this can beachieved by adjusting the concentrations and L/P of the detectionantibodies; (b) non-specific binding of detection antibodies to othercalibrators in the panel is also undesirable and should be minimized;(c) non-specific binding of other calibrators in the panel and otherrelated analytes should be minimized; if there is calibratornon-specific binding, it can reduce the overall specificity of theassays in the panel and it can also yield unreliable results as therewill be calibrator competition to bind the capture antibody.

In one specific embodiment, the kit further includes one or morereagents to insure that the correct steps of the assay protocol werefollowed. In addition, the variability of the assay steps can bemeasured independent of the assay. In this embodiment, a series ofcomplementary oligonucleotides diluted in diluents used to run an assayare provided in the kit. As the correct steps are performed, theoligonucleotides in the various diluents bind, extending the chain. Thefinal diluent can include the final complementary oligonucleotide with areporter or label to provide a detectable indicator of the successfulprocessing of the assay. For example, a first binding domain of amulti-well plate is coated with a BSA-oligonucleotide having sequence A.the assay diluent includes an oligonucleotide sequence complementary tosequence A, A′, and an additional sequence, B. A and B do not interactwith one another and specifically interact only with their complements.The final diluent contains a supplemental oligonucleotide comprising thecomplement of oligonucleotide B, B′, and a detectable label. Therefore,the detectable signal from the first binding domain can be used toverify that the sample was added or the detection moiety was added. Thesignal generated can also be used to detect if the correct volumes andconcentrations of reagents were added, as well as whether there wasvariability in sample handling procedures and/or equipment. Theoligonucleotide chain used in the process can include multipleoverlapping sequences.

Different assays in the panel may require different incubation times andsample handling requirements for optimal performance. Therefore, thegoal is to select a protocol that's optimized for most assays in thepanel. Optimization of the assay protocol includes, but is not limitedto, adjusting one or more of the following protocol parameters: timing(incubation time of each step), preparation procedure (calibrators,samples, controls, etc.), and number of wash steps.

In a further specific embodiment, the methods described in FIG. 5(c) canbe carried out using a kit configured for use in an instrument asdescribed in U.S. application Ser. No. 12/844,440, the disclosure ofwhich is incorporated herein by reference. The apparatus describedtherein enables fully automated random access analysis of samples usingarray-based multiplexed multi-well plate consumables. The apparatusachieves enhanced sensitivity and high sample throughput. All thebiological reagents required for an assay are added by the user and/orprovided in the apparatus prior to processing, thereby minimizing theconsumable and reagent requirements for the apparatus. Thus, the methodillustrated in FIG. 5(c) can be carried out in an apparatus as disclosedin U.S. Ser. No. 12/844,440 by providing the reagents required for themethod in a kit that includes a test plate, e.g., as illustrated in FIG.4(a)-(b) of U.S. Ser. No. 12/844,440, or as described herein as amulti-well assay plate, that includes the surface bearing targetingagent-modified binding domains, as well as (i) a set of one or morebinding reagents modified by supplemental linking agents, (ii) a set ofone or more targeting agent complements modified by linking agents;(iii) a set of detection reagents for the analytes of interest; andoptionally (iv) a set of control reagents and/or (v) a set ofcalibrators. The test plate can be mixed with kit components (i) and(ii), mixed with sample, and then detection reagents are added.Alternatively, components (i) and (ii) can be provided pre-mixed anddirectly combined with the surface, or components (i) and (ii) can beprovided in the kit as separate components that are mixed in solution bythe instrument and then combined with the surface as described above.Any surface binding step can optionally be followed by a washing step toremove any unbound components of the assay before proceeding to the nextstep. In a specific example, components (i) and (ii) are providedpre-mixed and added to the test plate in the instrument. The test plateis incubated, washed, the sample is optionally pre-diluted, and thenadded to the test plate. The test plate is incubated again, washed, anddetection reagents are added. The test plate is incubated once more,washed, read buffer is added, and signal is detected by the instrumentindicating the relative presence and/or absence of analyte(s) in thesample. The reagents can be provided in any suitable container in thekit, including test tubes or Eppendorf tubes, or in dedicated sectionsof an auxiliary plate as described in U.S. Ser. No. 12/844,440.

These and other embodiments of the invention are illustrated in thefollowing non-limited examples.

EXAMPLES Example 1—Direct Assay Format

The procedure for the preparation and use of a multi-well plate for adirect assay is illustrated in FIG. 2. The experimental layout, i.e.,which oligonucleotide sequence was located in which binding domain of amulti-well assay plate, was noted. The multi-well assay plate wasobtained from Meso Scale Discovery, a division of Meso ScaleDiagnostics, LLC (Rockville, Md.). A working solution of each individualoligonucleotide sequence complement (550 uL) was prepared by diluting astock solution of sequence complement about 50 times in Diluent 100(stock solutions of oligonucleotide sequence complement and Diluent 100are available from Meso Scale Discovery).

Each capture antibody was labeled with an oligonucleotide having aterminal thiol group using a bifunctional coupling reagent(sulfosuccinimidyl 4-(N-maléimidométhyl)-1-cyclohexane carboxylate(“SMCC”)) and conventional coupling protocols as shown in FIG. 5(d),e.g., protein is reacted with the NHS-ester in SMCC to label the proteinand the resulting complex is reacted with thiolated oligonucleotideswhich reacts with the maleimide group in SMCC. A pooled solution (50 uL)of a set of antibody-oligo-conjugates was then added to each well of themulti-well plate in hybridization buffer for 1 hour at room temperatureto hybridize the complementary oligonucleotide sequences and therebyimmobilize the capture antibodies to the multi-well plate to form aplurality of binding reagent complexes.

A solution including a plurality of analytes (25 uL of MSD Diluent 2,with 25 uL calibrator solution of MSD Diluent 2) was added to each wellof the prepared plate, incubated for 1 hour at room temperature, washed3×PBS, and a set of labeled detection antibodies (50 uL of MSD Diluent3) was added to each well of the multi-well plate. The plate wasincubated with shaking and the wells were washed with 3×PBS, filled with150 uL of Read Buffer T (Meso Scale Discovery) and analyzed on a SECTOR®Imager instrument.

This protocol was used to conduct an assay for a 7-plex chemokine paneland the result are shown in FIG. 8(a)-(g). The analytes assayed in thisexperiment were Eotaxin, MIP-1b, TARC, IL-8, MCP-4, IP-10, and MCP-1(all human analytes). In addition, this protocol was used to conduct anassay for a 10-plex TH1/TH2 panel including IFNg, IL-1b, IL-2, IL-4,IL-5, IL-8, IL-10, IL-12p70, IL-13, and TNFa (all human analytes). Theresults for both assays were compared with a standard direct immunoassayabsent oligonucleotides linkers.

Example 2—Indirect Assay Format Using an Oligonucleotide-SA Conjugate

The procedure for the preparation and use of a multi-well plate for anindirect assay using oligonucleotide-SA conjugates is illustrated inFIG. 9(a)-(c). The experimental layout for the multi-well assay platewas noted as in Example 1. A working solution of each individualoligonucleotide sequence complement bound to streptavidin (SA) (550 uL)was prepared by diluting a stock solution of sequence complement about50 times in Diluent 100 (stock solutions of oligonucleotide sequencecomplement and Diluent 100 are available from Meso Scale Discovery). Asolution of biotinylated antibody was added to the desired workingsolution of oligonucleotide sequence complement to prepare a set ofindividual biotinylated capture antibody/oligonucleotide-SA mixtures.The concentration of biotinylated antibody in the mixture was in therange of about 5-30 ug/mL. The mixture was gently mixed for 30-45minutes at room temperature. Fifty (50) uL of biotin solution (MesoScale Discovery) (approximately a three-fold excess of biotin) was addedto each individual biotinylated antibody/oligonucleotide complementmixture and this mixture was gently mixed for 10-15 minutes at roomtemperature. Equal volumes (550 uL) of individual biotinylatedantibody/oligonucleotide complements were combined and the total volumeof the solution was adjusted to 5500 uL with the addition of aConjugation Buffer (PBS with 0.1 M EDTA at pH 7.4).

The multi-well plate was allowed to warm to room temperature(approximately 10 minutes). Fifty (50) uL of biotinylatedantibodies/oligonucleotide complements was added to each well of theplate. The plate was covered with an adhesive seal and incubated for 1hour on a plate shaker at room temperature. Each well was washed withphosphate buffered saline (PBS, 3×).

A solution including a plurality of analytes (25 uL of MSD Diluent 2,with 25 uL calibrator solution of MSD Diluent 2) was added to each wellof the prepared plate, incubated for 1 hour at room temperature, washed3×PBS, and a set of labeled detection antibodies (50 uL of MSD Diluent3) was added to each well of the multi-well plate. The plate wasincubated with shaking and the wells were washed with 3×PBS, filled with150 uL of Read Buffer T (Meso Scale Discovery) and analyzed on a SECTOR®Imager instrument.

Example 3—Indirect Assay Format Using Neat SA/BiotinylatedOligonucleotides

The procedure for the preparation and use of a multi-well plate for anindirect assay using biotinylated capture antibodies, neat streptavidin,and biotinylated oligonucleotides is illustrated in FIG. 10(a)-(c). Theexperimental layout for the multi-well assay plate was noted as inExample 1. A working solution of an excess of each individualoligonucleotide sequence complement bound to streptavidin (SA) (550 uL)was prepared by diluting a stock solution of sequence complement about50 times in Diluent 100 (stock solutions of oligonucleotide sequencecomplement and Diluent 100 are available from Meso Scale Discovery). Asolution of biotinylated antibody was added to the desired workingsolution of oligonucleotide sequence complement to prepare a set ofindividual biotinylated capture antibody/oligonucleotide-SA mixtures.The concentration of biotinylated antibody in the mixture was in therange of about 5-30 ug/mL. The mixture was gently mixed for 30-45minutes at room temperature. Fifty (50) uL of biotin solution (MesoScale Discovery) (approximately a three-fold excess of biotin) was addedto each individual biotinylated antibody/oligonucleotide complementmixture and this mixture was gently mixed for 10-15 minutes at roomtemperature. Equal volumes (550 uL) of individual biotinylatedantibody/oligonucleotide complements were combined and the total volumeof the solution was adjusted to 5500 uL with the addition of aConjugation Buffer (NEED COMPOSITION; available from Meso ScaleDiscovery).

The multi-well plate was allowed to warm to room temperature(approximately 10 minutes). Fifty (50) uL of biotinylatedantibodies/oligonucleotide complements was added to each well of theplate. The plate was covered with an adhesive seal and incubated for 1hour on a plate shaker at room temperature. Each well was washed withphosphate buffered saline (PBS, 3×).

A solution including a plurality of analytes (25 uL of MSD Diluent 2,with 25 uL calibrator solution of MSD Diluent 2) was added to each wellof the prepared plate, incubated for 1 hour at room temperature, washed3×PBS, and a set of labeled detection antibodies (50 uL of MSD Diluent3) was added to each well of the multi-well plate. The plate wasincubated with shaking and the wells were washed with 3×PBS, filled with150 uL of Read Buffer T (Meso Scale Discovery) and analyzed on a SECTOR®Imager instrument.

Example 4—Comparative Results of Three Assay Formats Using 7-PlexCytokine B Panel

A 7-plex cytokine B panel (IL-8, hTNF-a, hEotaxin-3, h-Eotaxin, hMCP-1,HIP-10 and hMIP-1a) was tested in the assay formats described inExamples 1, 2, and 3, i.e. direct, indirect with oligonucleotide-SAconjugate and indirect with neat SA/biotinylated oligonucleotides. LODvalues for assays were estimated from an 8-point calibration curveassume 2.5 standard deviations. The results are shown in FIG. 11(a)-(g).The LOD values for direct and indirect assay formats compare with LODvalues observed for standard passive adsorption and immunoassay formatsfor all tested assays. The indirect approach with neat SA showedsignificant spread in LOD values: some assays, IL-8 and hIP-10, showedhigher LOD values compared to the remaining assay formats, while Eotaxinand MIP-10 showed lower LOD values.

The present invention is not to be limited in scope by the specificembodiments described herein. Indeed, various modifications of themethod in addition to those described herein will become apparent tothose skilled in the art from the foregoing description and accompanyingfigures. Such modifications are intended to fall within the scope of theclaims. Various publications are cited herein, the disclosures of whichare incorporated by reference in their entireties.

The invention claimed is:
 1. A method of conducting a multiplexedbinding assay for a plurality of analytes of interest comprising (a)combining, in one or more steps, the following components: (i) a samplecomprising a first analyte of interest and a second analyte of interest,(ii) a first targeting agent immobilized on a first binding domain,(iii) a first targeting agent complement connected to a linking agent,wherein the first targeting agent complement is a binding partner of thefirst targeting agent, (iv) a first binding reagent connected to asupplemental linking agent, wherein the first binding reagent is abinding partner of the first analyte, (v) a second targeting agentimmobilized on a second binding domain, (vi) a second targeting agentcomplement connected to a linking agent, wherein the second targetingagent complement is a binding partner of the second targeting agent,(vii) a second binding reagent connected to a supplemental linkingagent, wherein the second binding reagent is a binding partner of thesecond analyte, and (viii) optionally, at least two copies of a bridgingagent, wherein, if the bridging agent is omitted, each linking agent isa binding partner of the supplemental linking agent, or if the bridgingagent is included, the bridging agent has a first binding site for oneof the linking agents and an additional binding site for one of thesupplemental linking agents; (b) forming (i) a first binding complex onthe first binding domain comprising the first targeting agent, the firsttargeting agent complement, the first binding reagent and the firstanalyte, and (ii) a second binding complex on the second binding domaincomprising the second targeting agent, the second targeting agentcomplement, the second binding reagent and the second analyte, and (c)measuring the amount of the first and second analytes on the first andsecond binding domains, respectively wherein the complementaryoligonucleotide pair positioned on each of the first and second bindingdomains is different and selected from: Pair Sequence 1acatcggtagtt (SEQ ID NO: 1) aactaccgatgt (SEQ ID NO: 2) 2acgtcccagttg (SEQ ID NO: 3) caactgggacgt (SEQ ID NO: 4) 3agaagaagatcc (SEQ ID NO: 5) ggatcttcttct (SEQ ID NO: 6) 4aggttcaftgca (SEQ ID NO: 7) tgcactgaacct (SEQ ID NO: 8) 5atcaggatacgc (SEQ ID NO: 9) gcgtatcctgat (SEQ ID NO: 10) 6atcattaccacc (SEQ ID NO: 11) ggtggtaatgat (SEQ ID NO: 12) 7attaacgggagc (SEQ ID NO: 13) gctcccgttaat (SEQ ID NO: 14) 8cagaggtcttaa (SEQ ID NO: 15) ttaagacctctg (SEQ ID NO: 16) 9caggtgtccatt (SEQ ID NO: 17) aatggacacctg (SEQ ID NO: 18) 10catccaatccag (SEQ ID NO: 19) ctggattggatg (SEQ ID NO: 20) 11cctacgatatac (SEQ ID NO: 21) gtatatcgtagg (SEQ ID NO: 22) 12cgaatgtagagt (SEQ ID NO: 23) actctacattcg (SEQ ID NO: 24) 13cggtttgagata (SEQ ID NO: 25) tatctcaaaccg (SEQ ID NO: 26) 14cttacaacgcca (SEQ ID NO: 27) tggcgttgtaag (SEQ ID NO: 28) 15ctttctcggcac (SEQ ID NO: 29) gtgccgagaaag (SEQ ID NO: 30) 16gacataaagcga (SEQ ID NO: 31) tcgctttatgtc (SEQ ID NO: 32) 17gccatagtctct (SEQ ID NO: 33) agagactatggc (SEQ ID NO: 34) 18gctaattcacca (SEQ ID NO: 35) tggtgaattagc (SEQ ID NO: 36) 19ggtcgtgtttca (SEQ ID NO: 37) tgaaacacgacc (SEQ ID NO: 38) 20gttgattctgtc (SEQ ID NO: 39) gacagaatcaac (SEQ ID NO: 40) 23ttccacttaggg (SEQ ID NO: 45) ccctaagtggaa (SEQ ID NO: 46) 25tttcccttgcta (SEQ ID NO: 49) tagcaagggaaa (SEQ ID NO: 50)

wherein each of SEQ ID NOs: 1, 5, 11, 21 and 25 are modified with alinking agent comprising a thiol group and each of SEQ ID NOs: 2, 6, 12,22 and 26 are modified with a linking agent comprising a biotin group.2. The method of claim 1, wherein (i) the first targeting agent andfirst targeting agent complement and (ii) the second targeting agent andsecond targeting agent complement each comprise a complementaryoligonucleotide pair.
 3. The method of claim 2, wherein thecomplementary oligonucleotide pair positioned on each of the first andsecond binding domains is different and selected from: Pair Sequence 1acatcggtagtt (SEQ ID NO: 1) aactaccgatgt (SEQ ID NO: 2) 3agaagaagatcc (SEQ ID NO: 5) ggatcttcttct (SEQ ID NO: 6) 6atcattaccacc (SEQ ID NO: 11) ggtggtaatgat (SEQ ID NO: 12) 11cctacgatatac (SEQ ID NO: 21) gtatatcgtagg (SEQ ID NO: 22) 13cggtttgagata (SEQ ID NO: 25) tatctcaaaccg (SEQ ID NO: 26) 16gacataaagcga (SEQ ID NO: 31) tcgctttatgtc (SEQ ID NO: 32) 17gccatagtctct (SEQ ID NO: 33) agagactatggc (SEQ ID NO: 34) 18gctaattcacca (SEQ ID NO: 35) tggtgaattagc (SEQ ID NO: 36) 20gttgattctgtc (SEQ ID NO: 39) gacagaatcaac (SEQ ID NO: 40) 25tttcccttgcta (SEQ ID NO: 49) tagcaagggaaa (SEQ ID NO: 50)

wherein each of SEQ ID NOs: 1, 5, 11, 21 and 25 are modified with alinking agent comprising a thiol group and each of SEQ ID NOs: 2, 6, 12,22 and 26 are modified with a linking agent comprising a biotin group.4. The method of claim 2, wherein the complementary oligonucleotide pairpositioned on each of the first and second binding domains is differentand selected from: Pair Sequence 2 acgtcccagttg (SEQ ID NO: 3)caactgggacgt (SEQ ID NO: 4) 4 aggttcaftgca (SEQ ID NO: 7)tgcactgaacct (SEQ ID NO: 8) 5 atcaggatacgc (SEQ ID NO: 9)gcgtatcctgat (SEQ ID NO: 10) 8 cagaggtcttaa (SEQ ID NO: 15)ttaagacctctg (SEQ ID NO: 16) 10 catccaatccag (SEQ ID NO: 19)ctggattggatg (SEQ ID NO: 20) 12 cgaatgtagagt (SEQ ID NO: 23)actctacattcg (SEQ ID NO: 24) 14 cttacaacgcca (SEQ ID NO: 27)tggcgttgtaag (SEQ ID NO: 28) 15 ctttctcggcac (SEQ ID NO: 29)gtgccgagaaag (SEQ ID NO: 30) 19 ggtcgtgtttca (SEQ ID NO: 37)tgaaacacgacc (SEQ ID NO: 38) 23 ttccacttaggg (SEQ ID NO: 45)ccctaagtggaa (SEQ ID NO: 46).


5. The method of claim 2, wherein the complementary oligonucleotide pairpositioned on each of the first and second binding domains is differentand selected from: Pair Sequence 2 acgtcccagttg (SEQ ID NO: 3)caactgggacgt (SEQ ID NO: 4) 4 aggttcaftgca (SEQ ID NO: 7)tgcactgaacct (SEQ ID NO: 8) 5 atcaggatacgc (SEQ ID NO: 9)gcgtatcctgat (SEQ ID NO: 10) 7 attaacgggagc (SEQ ID NO: 13)gctcccgttaat (SEQ ID NO: 14) 8 cagaggtcttaa (SEQ ID NO: 15)ttaagacctctg (SEQ ID NO: 16) 9 caggtgtccatt (SEQ ID NO: 17)aatggacacctg (SEQ ID NO: 18) 10 catccaatccag (SEQ ID NO: 19)ctggattggatg (SEQ ID NO: 20) 12 cgaatgtagagt (SEQ ID NO: 23)actctacattcg (SEQ ID NO: 24) 14 cttacaacgcca (SEQ ID NO: 27)tggcgttgtaag (SEQ ID NO: 28) 23 ttccacttaggg (SEQ ID NO: 45)ccctaagtggaa (SEQ ID NO: 46).


6. The method of claim 2, wherein the complementary oligonucleotide pairpositioned on each of the first and second binding domains is differentand selected from: Pair Sequence 1 acatcggtagtt (SEQ ID NO: 1)aactaccgatgt (SEQ ID NO: 2) 3 agaagaagatcc (SEQ ID NO: 5)ggatcttcttct (SEQ ID NO: 6) 7 attaacgggagc (SEQ ID NO: 13)gctcccgttaat (SEQ ID NO: 14) 8 cagaggtcttaa (SEQ ID NO: 15)ttaagacctctg (SEQ ID NO: 16) 9 caggtgtccatt (SEQ ID NO: 17)aatggacacctg (SEQ ID NO: 18) 12 cgaatgtagagt (SEQ ID NO: 23)actctacattcg (SEQ ID NO: 24) 17 gccatagtctct (SEQ ID NO: 33)agagactatggc (SEQ ID NO: 34) 18 gctaattcacca (SEQ ID NO: 35)tggtgaattagc (SEQ ID NO: 36) 23 ttccacttaggg (SEQ ID NO: 45)ccctaagtggaa (SEQ ID NO: 46) 25 tttcccttgcta (SEQ ID NO: 49)tagcaagggaaa (SEQ ID NO: 50).


7. The method of claim 2, wherein the complementary oligonucleotide pairpositioned on each of the first and second binding domains is differentand selected from: Pair Sequence 5 atcaggatacgc (SEQ ID NO: 9)gcgtatcctgat (SEQ ID NO: 10) 6 atcattaccacc (SEQ ID NO: 11)ggtggtaatgat (SEQ ID NO: 12) 10 catccaatccag (SEQ ID NO: 19)ctggattggatg (SEQ ID NO: 20) 11 cctacgatatac (SEQ ID NO: 21)gtatatcgtagg (SEQ ID NO: 22) 13 cggtttgagata (SEQ ID NO: 25)tatctcaaaccg (SEQ ID NO: 26) 14 cttacaacgcca (SEQ ID NO: 27)tggcgttgtaag (SEQ ID NO: 28) 15 ctttctcggcac (SEQ ID NO: 29)gtgccgagaaag (SEQ ID NO: 30) 16 gacataaagcga (SEQ ID NO: 31)tcgctttatgtc (SEQ ID NO: 32) 19 ggtcgtgtttca (SEQ ID NO: 37)tgaaacacgacc (SEQ ID NO: 38) 20 gttgattctgtc (SEQ ID NO: 39)gacagaatcaac (SEQ ID NO: 40).


8. The method of claim 2, wherein the complementary oligonucleotide pairpositioned on each of the first and second binding domains is differentand selected from: Pair Sequence 1 acatcggtagtt (SEQ ID NO: 1)aactaccgatgt (SEQ ID NO: 2) 2 acgtcccagttg (SEQ ID NO: 3)caactgggacgt (SEQ ID NO: 4) 3 agaagaagatcc (SEQ ID NO: 5)ggatcttcttct (SEQ ID NO: 6) 4 aggttcaftgca (SEQ ID NO: 7)tgcactgaacct (SEQ ID NO: 8) 6 atcattaccacc (SEQ ID NO: 11)ggtggtaatgat (SEQ ID NO: 12) 9 caggtgtccatt (SEQ ID NO: 17)aatggacacctg (SEQ ID NO: 18) 11 cctacgatatac (SEQ ID NO: 21)gtatatcgtagg (SEQ ID NO: 22) 12 cgaatgtagagt (SEQ ID NO: 23)actctacattcg (SEQ ID NO: 24) 16 gacataaagcga (SEQ ID NO: 31)tcgctttatgtc (SEQ ID NO: 32) 20 gttgattctgtc (SEQ ID NO: 39)gacagaatcaac (SEQ ID NO: 40).


9. The method of claim 2, wherein the complementary oligonucleotide pairpositioned on each of the first and second binding domains is differentand selected from: Pair Sequence 3 agaagaagatcc (SEQ ID NO: 5)ggatcttcttct (SEQ ID NO: 6) 5 atcaggatacgc (SEQ ID NO: 9)gcgtatcctgat (SEQ ID NO: 10) 7 attaacgggagc (SEQ ID NO: 13)gctcccgttaat (SEQ ID NO: 14) 8 cagaggtcttaa (SEQ ID NO: 15)ttaagacctctg (SEQ ID NO: 16) 9 caggtgtccatt (SEQ ID NO: 17)aatggacacctg (SEQ ID NO: 18) 10 catccaatccag (SEQ ID NO: 19)ctggattggatg (SEQ ID NO: 20) 13 cggtttgagata (SEQ ID NO: 25)tatctcaaaccg (SEQ ID NO: 26) 16 gacataaagcga (SEQ ID NO: 31)tcgctttatgtc (SEQ ID NO: 32) 23 ttccacttaggg (SEQ ID NO: 45)ccctaagtggaa (SEQ ID NO: 46) 25 tttcccttgcta (SEQ ID NO: 49)tagcaagggaaa (SEQ ID NO: 50).


10. The method of claim 2, wherein the complementary oligonucleotidepair positioned on each of the first and second binding domains isdifferent and selected from: Pair Sequence 1 acatcggtagtt (SEQ ID NO: 1)aactaccgatgt (SEQ ID NO: 2) 2 acgtcccagttg (SEQ ID NO: 3)caactgggacgt (SEQ ID NO: 4) 3 agaagaagatcc (SEQ ID NO: 5)ggatcttcttct (SEQ ID NO: 6) 4 aggttcaftgca (SEQ ID NO: 7)tgcactgaacct (SEQ ID NO: 8) 5 atcaggatacgc (SEQ ID NO: 9)gcgtatcctgat (SEQ ID NO: 10) 10 catccaatccag (SEQ ID NO: 19)ctggattggatg (SEQ ID NO: 20) 14 cttacaacgcca (SEQ ID NO: 27)tggcgttgtaag (SEQ ID NO: 28) 17 gccatagtctct (SEQ ID NO: 33)agagactatggc (SEQ ID NO: 34) 18 gctaattcacca (SEQ ID NO: 35)tggtgaattagc (SEQ ID NO: 36) 25 tttcccttgcta (SEQ ID NO: 49)tagcaagggaaa (SEQ ID NO: 50).


11. The method of claim 2, wherein the complementary oligonucleotidepair positioned on each of the first and second binding domains isdifferent and selected from: Pair Sequence 5 atcaggatacgc (SEQ ID NO: 9)gcgtatcctgat (SEQ ID NO: 10) 7 attaacgggagc (SEQ ID NO: 13)gctcccgttaat (SEQ ID NO: 14) 8 cagaggtcttaa (SEQ ID NO: 15)ttaagacctctg (SEQ ID NO: 16) 9 caggtgtccatt (SEQ ID NO: 17)aatggacacctg (SEQ ID NO: 18) 10 catccaatccag (SEQ ID NO: 19)ctggattggatg (SEQ ID NO: 20) 12 cgaatgtagagt (SEQ ID NO: 23)actctacattcg (SEQ ID NO: 24) 14 cttacaacgcca (SEQ ID NO: 27)tggcgttgtaag (SEQ ID NO: 28) 15 ctttctcggcac (SEQ ID NO: 29)gtgccgagaaag (SEQ ID NO: 30) 19 ggtcgtgtttca (SEQ ID NO: 37)tgaaacacgacc (SEQ ID NO: 38) 23 ttccacttaggg (SEQ ID NO: 45)ccctaagtggaa (SEQ ID NO: 46).


12. The method of claim 2, wherein the complementary oligonucleotidepair positioned on each of the first and second binding domains isdifferent and selected from: Pair Sequence 2 acgtcccagttg (SEQ ID NO: 3)caactgggacgt (SEQ ID NO: 4) 3 agaagaagatcc (SEQ ID NO: 5)ggatcttcttct (SEQ ID NO: 6) 4 aggttcaftgca (SEQ ID NO: 7)tgcactgaacct (SEQ ID NO: 8) 5 atcaggatacgc (SEQ ID NO: 9)gcgtatcctgat (SEQ ID NO: 10) 8 cagaggtcttaa (SEQ ID NO: 15)ttaagacctctg (SEQ ID NO: 16) 16 gacataaagcga (SEQ ID NO: 31)tcgctttatgtc (SEQ ID NO: 32) 18 gctaattcacca (SEQ ID NO: 35)tggtgaattagc (SEQ ID NO: 36) 20 gttgattctgtc (SEQ ID NO: 39)gacagaatcaac (SEQ ID NO: 40) 23 ttccacttaggg (SEQ ID NO: 45)ccctaagtggaa (SEQ ID NO: 46) 25 tttcccttgcta (SEQ ID NO: 49)tagcaagggaaa (SEQ ID NO: 50).


13. The method of claim 1, wherein the measuring step (c) furthercomprises measuring the presence of the first and second detectablelabels via optical absorbance, fluorescence, phosphorescence,chemiluminescence, electrochemiluminescence, light scattering, ormagnetism.
 14. The method of claim 13, wherein the first and seconddetectable label is an electrochemiluminescent label and the measuringstep (c) further comprises measuring an electrochemiluminescent signaland correlating the signal with an amount of first and second analyte inthe sample.
 15. The method of claim 1, wherein the first and secondbinding domains are positioned on an electrode and the measuring stepfurther comprises applying a voltage waveform to the electrode togenerate electrochemiluminescence.
 16. The method of claim 1, whereineach of the first and second binding domains is an element of an arrayof binding domains.
 17. The method of claim 16, wherein the array islocated within a well of a multi-well plate.
 18. The method of claim 1,wherein each of the first and second binding domains are each positionedon a surface of one or more particles.
 19. The method of claim 18,wherein the particles are coded to allow for identification of specificparticles and discrimination between the first and second bindingdomains.